阅读说明：本技术 分解含反式脂肪酸油脂的新型脂肪酶 (Novel lipase for decomposing trans-fatty acid-containing oil ) 是由 堀克敏 于 2019-07-05 设计创作，主要内容包括：本公开提供分解含反式脂肪酸油脂的技术。本公开的新型脂肪酶具有分解含反式脂肪酸油脂的活性。本公开的脂肪酶包含：由序列号4、11、16或18所示的氨基酸序列构成的多肽、或由序列号1、7、15或17的核酸序列编码的多肽、或与它们具有至少70％的序列同源性的多肽。发现该脂肪酶在高温下也具有分解油脂的能力,热稳定性优异。本公开的脂肪酶例如在排水处理、清洗剂、脂肪改性/油脂生产技术等油处理中是有用的。(The present disclosure provides a technique for decomposing trans-fatty acid-containing oils and fats. The novel lipase of the present disclosure has an activity of decomposing trans-fatty acid-containing oils and fats. The lipase of the present disclosure comprises: a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 4, 11, 16 or 18, or a polypeptide encoded by the nucleic acid sequence of SEQ ID NO.1, 7, 15 or 17, or a polypeptide having at least 70% sequence homology thereto. The lipase was found to have an ability to decompose fats and oils even at high temperatures and to have excellent thermal stability. The lipase of the present disclosure is useful, for example, in oil treatment such as drainage treatment, detergent, fat modification/oil production technology, and the like.)

1. A polypeptide which is:

(a) a polypeptide comprising the amino acid sequence shown in seq id No. 4, 11, 16 or 18;

(b) a polypeptide comprising an amino acid sequence comprising substitution, addition, deletion, or combination thereof of 1 or more amino acids in the amino acid sequence of (a) and having biological activity;

(c) a polypeptide having a sequence identity of at least 70% or more with the amino acid sequence represented by (a) or (b) and having a biological activity;

(d) a polypeptide comprising an amino acid sequence encoded by the nucleic acid sequence set forth in seq id No.1, 7, 15, or 17;

(e) a polypeptide having biological activity encoded by a nucleic acid sequence comprising substitution, addition, deletion or combination thereof of 1 or more nucleotides in the nucleic acid sequence shown in (d);

(f) a polypeptide encoded by a nucleic acid sequence having at least 70% or more sequence identity to the nucleic acid sequence of (d) or (e) and having biological activity;

(g) a polypeptide having a biological activity encoded by a nucleic acid sequence that hybridizes under stringent conditions to a polynucleotide comprising the nucleic acid sequence shown in any one of (d) to (f) or a complementary sequence thereof;

(h) a polypeptide encoded by an allelic variant of any of the nucleic acid sequences of (d) to (g) and having biological activity; or

(i) A polypeptide comprising a fragment of the amino acid sequence shown in (a) to (h).

2. The polypeptide according to claim 1, wherein the biological activity is an ability to assimilate a trans-fatty acid-containing oil or fat or an ability to decompose a trans-fatty acid-containing oil or fat.

3. The polypeptide according to claim 1 or 2, wherein 45 to 70 ℃ is an optimum temperature for the ability to decompose oils and fats.

4. The polypeptide according to claim 1 or 2, wherein 35 to 55 ℃ is an optimum temperature for the ability to decompose oils and fats.

5. The polypeptide according to any one of claims 1 to 3, which is thermostable at 65 ℃ or higher.

6. The polypeptide of any one of claims 1, 2 or 4, which is thermostable above 75 ℃.

7. The polypeptide according to any one of claims 1 to 6, which has an ability to decompose fats and oils at 15 ℃.

8. The polypeptide of any one of claims 1 to 7, which is a polypeptide from Burkholderia forestea (Burkholderia arboris).

9. A polynucleotide which is:

(A) a polynucleotide comprising the nucleic acid sequence set forth in seq id No.1, 7, 15, or 17;

(B) a polynucleotide comprising a nucleic acid sequence comprising substitution, addition, deletion or combination thereof of 1 or more nucleotides in the nucleic acid sequence shown in (A);

(C) a polynucleotide comprising a nucleic acid sequence having at least 70% or more sequence identity to the nucleic acid sequence of (A) or (B) and encoding a polypeptide having biological activity;

(D) a polynucleotide encoding a polypeptide having a biological activity, the polynucleotide comprising a nucleic acid sequence that hybridizes under stringent conditions to a polynucleotide comprising the nucleic acid sequence shown in any one of (A) to (C) or a complementary sequence thereof;

(E) a polynucleotide encoding a polypeptide having a biological activity which is an allelic variant of any of the nucleic acid sequences (A) to (D); or

(F) A polynucleotide encoding a polypeptide comprising the amino acid sequence shown in seq id No. 4, 11, 16 or 18;

(G) a polynucleotide encoding a polypeptide having a biological activity, which comprises an amino acid sequence comprising substitution, addition, deletion or combination of 1 or more amino acids in the amino acid sequence of (F);

(H) a polynucleotide encoding a polypeptide having a sequence identity of at least 70% or more to the amino acid sequence represented by (F) or (G) and having a biological activity; or

(I) A polynucleotide comprising a fragment of the nucleic acid sequence shown in (F) to (H).

10. The polynucleotide according to claim 9, wherein said biological activity is an ability to assimilate a trans-fatty acid-containing oil or fat or an ability to decompose a trans-fatty acid-containing oil or fat.

11. The polynucleotide according to claim 9 or 10, which encodes a polypeptide having an optimum temperature for the ability to decompose oils and fats at 45 to 70 ℃.

12. The polynucleotide according to claim 9 or 10, which encodes a polypeptide having an optimum temperature for the ability to decompose oils and fats at 35 to 55 ℃.

13. A polynucleotide according to any one of claims 9 to 11 which encodes a polypeptide which is thermostable at 65 ℃ or higher.

14. The polynucleotide of any one of claims 9, 10, or 12, which encodes a polypeptide that is thermostable at 75 ℃ or higher.

15. The polynucleotide according to any one of claims 9 to 14, wherein the polynucleotide encodes a polypeptide having an ability to decompose oils and fats at 15 ℃.

16. The polynucleotide of any one of claims 9-15, which is a polynucleotide from Burkholderia forestea (Burkholderia arboris).

17. A cell or cell-free expression system comprising the polynucleotide of any one of claims 9-16.

18. An oil breakdown agent comprising: a polypeptide according to any one of claims 1 to 8, or a cellular or cell-free expression system comprising a polynucleotide according to any one of claims 9 to 16, or a cellular or cell-free expression system according to claim 17.

19. An oil breakdown agent according to claim 18, further comprising an oil treatment ingredient.

20. A kit for decomposing oil, comprising: a polypeptide according to any one of claims 1 to 8, or a cell or cell-free expression system comprising a polynucleotide according to any one of claims 9 to 16, or a cell or cell-free expression system according to claim 17, or an oil breakdown agent according to claim 18; and further oil treatment components.

21. An oil breakdown removal method comprising: allowing the polypeptide of any one of claims 1 to 8, or a cell or cell-free expression system comprising the polynucleotide of any one of claims 9 to 16, the cell or cell-free expression system of claim 17, or the oil-solubilizing agent of claim 18 or 19 to act on a subject.

22. The method according to claim 21, wherein the treatment object contains trans fatty acid or trans fatty acid-containing fat.

23. A cleaning formulation, comprising: a polypeptide according to any one of claims 1 to 8, or a cellular or cell-free expression system comprising a polynucleotide according to any one of claims 9 to 16, or a cellular or cell-free expression system according to claim 17.

24. Use of a polypeptide according to any one of claims 1 to 8, or a cell or cell-free expression system comprising a polynucleotide according to any one of claims 9 to 16, a cell or cell-free expression system according to claim 17, or an oil breakdown agent according to claim 18 or 19 in fat modification/oil production technology.

Technical Field

The present disclosure relates to a novel lipase for decomposing trans-fatty acid-containing oils and fats, and applications thereof (e.g., drainage treatment, oil treatment). In another aspect, the present disclosure relates to novel lipases and uses thereof that are capable of decomposing lipids also at high temperatures.

Background

As typical examples of the dirt generated in general households, catering industry, industrial equipment, and the like, oil dirt can be cited. Grease dirt is dirt that is difficult to wash and is generated in a kitchen, a sink, a kitchen, a pipeline, a drain, a ventilation fan, washing, and the like. Oil stains are a source of offensive odor and vermin and may cause environmental pollution, and thus establishment of a revolutionary technique in oil treatment is urgently desired from both public health and the environment.

In view of the fact that a grease trap, which is a treatment device for removing oil contained in kitchen wastewater in the catering industry by solid-liquid separation, is a source of generation of offensive odor and harmful insects, and that it is necessary to take physical effort and cost for recovery, transportation, cleaning, and other maintenance of the separated oil, a revolutionary technique for eliminating the oil in the grease trap has been desired in the catering industry, and the like.

Disclosure of Invention

Means for solving the problems

As a result of intensive studies, the present inventors have found a lipase which decomposes trans-fatty acid-containing oils and fats. The lipase was also found to have an ability to decompose fats and oils even at high temperatures and to have excellent thermal stability. The disclosure also relates to uses of the lipases of the disclosure, such as oil treatment and the like.

Accordingly, the present disclosure provides the following.

(item 1)

A polypeptide which is:

(a) a polypeptide comprising the amino acid sequence shown in seq id No. 4, 11, 16 or 18;

(b) a polypeptide comprising an amino acid sequence comprising substitution, addition, deletion, or combination thereof of 1 or more amino acids in the amino acid sequence of (a) and having biological activity;

(c) a polypeptide having a sequence identity of at least 70% or more with the amino acid sequence represented by (a) or (b) and having a biological activity;

(d) a polypeptide comprising an amino acid sequence encoded by the nucleic acid sequence set forth in seq id No.1, 7, 15, or 17;

(e) a polypeptide having biological activity encoded by a nucleic acid sequence comprising substitution, addition, deletion or combination thereof of 1 or more nucleotides in the nucleic acid sequence shown in (d);

(f) a polypeptide encoded by a nucleic acid sequence having at least 70% or more sequence identity to the nucleic acid sequence of (d) or (e) and having biological activity;

(g) a polypeptide having a biological activity encoded by a nucleic acid sequence that hybridizes under stringent conditions to a polynucleotide comprising the nucleic acid sequence shown in any one of (d) to (f) or a complementary sequence thereof;

(h) a polypeptide encoded by an allelic variant of any of the nucleic acid sequences of (d) to (g) and having biological activity; or

(i) A polypeptide comprising a fragment of the amino acid sequence shown in (a) to (h).

(item 2)

The polypeptide according to item 1, wherein the biological activity is an ability to assimilate a trans-fatty acid-containing oil or fat or an ability to decompose a trans-fatty acid-containing oil or fat.

(item 3)

The polypeptide according to item 1 or 2, wherein 45 to 70 ℃ is an optimum temperature for the ability to decompose oil or fat.

(item 4)

The polypeptide according to any one of items 1 to 3, wherein 35 to 55 ℃ is an optimum temperature for the ability to decompose an oil or fat.

(item 5)

The polypeptide according to any one of items 1 to 4, which has thermostability at 65 ℃ or more and preferably 85 ℃ or less.

(item 6)

The polypeptide according to any one of items 1 to 5, which has thermostability at 75 ℃ or more and preferably 80 ℃ or less.

(item 7)

The polypeptide according to any one of items 1 to 6, which has an ability to decompose oils and fats at 15 ℃.

(item 8)

The polypeptide according to any one of items 1 to 7, which is a polypeptide from Burkholderia forestea (Burkholderia arboris).

(item 9)

A polynucleotide which is:

(A) a polynucleotide comprising the nucleic acid sequence set forth in seq id No.1, 7, 15, or 17;

(B) a polynucleotide comprising a nucleic acid sequence comprising substitution, addition, deletion or combination thereof of 1 or more nucleotides in the nucleic acid sequence shown in (A);

(C) a polynucleotide comprising a nucleic acid sequence having at least 70% or more sequence identity to the nucleic acid sequence of (A) or (B) and encoding a polypeptide having biological activity;

(D) a polynucleotide encoding a polypeptide having a biological activity, the polynucleotide comprising a nucleic acid sequence that hybridizes under stringent conditions to a polynucleotide comprising the nucleic acid sequence shown in any one of (A) to (C) or a complementary sequence thereof;

(E) a polynucleotide encoding a polypeptide having a biological activity which is an allelic variant of any of the nucleic acid sequences (A) to (D); or

(F) A polynucleotide encoding a polypeptide comprising the amino acid sequence shown in seq id No. 4, 11, 16 or 18;

(G) a polynucleotide encoding a polypeptide having a biological activity, which comprises an amino acid sequence comprising substitution, addition, deletion or combination of 1 or more amino acids in the amino acid sequence of (F);

(H) a polynucleotide encoding a polypeptide having a sequence identity of at least 70% or more to the amino acid sequence represented by (F) or (G) and having a biological activity; or

(I) A polynucleotide comprising a fragment of the nucleic acid sequence shown in (F) to (H).

(item 10)

The polynucleotide according to item 9, wherein the biological activity is an ability to assimilate a trans-fatty acid-containing oil or fat or an ability to decompose a trans-fatty acid-containing oil or fat.

(item 11)

The polynucleotide according to item 9 or 10, which encodes a polypeptide having an optimum temperature for the ability to decompose oils and fats at 45 to 70 ℃.

(item 12)

The polynucleotide according to any one of items 9 to 11, which encodes a polypeptide having an optimum temperature for the ability to decompose oils and fats at 35 to 55 ℃.

(item 13)

A polynucleotide according to any one of items 9 to 12 which encodes a polypeptide which is thermostable at 65 ℃ or more and preferably 85 ℃ or less.

(item 14)

A polynucleotide according to any one of items 9 to 13 which encodes a polypeptide which is thermostable at 75 ℃ or more and preferably 80 ℃ or less.

(item 15)

The polynucleotide according to any one of items 9 to 14, wherein the polynucleotide encodes a polypeptide having an ability to decompose oils and fats at 15 ℃.

(item 16)

The polynucleotide according to any one of items 9 to 15, which is a polynucleotide from Burkholderia forestea (Burkholderia arboris).

(item 17)

A cell or cell-free expression system comprising the polynucleotide of any one of items 9-16.

(item 18)

An oil breakdown agent comprising: a polypeptide according to any one of items 1 to 8, a cell or cell-free expression system comprising a polynucleotide according to any one of items 9 to 16, or a cell or cell-free expression system according to item 17.

(item 19)

The oil breakdown agent of clause 18, further comprising an oil treatment ingredient.

(item 20)

A kit for decomposing oil, comprising: a polypeptide of any one of items 1 to 8, or a cell or cell-free expression system comprising a polynucleotide of any one of items 9 to 16, or a cell or cell-free expression system of item 17, or an oil breakdown agent of item 18; and further oil treatment components.

(item 21)

An oil breakdown removal method comprising: a polypeptide according to any one of items 1 to 8, or a cell or cell-free expression system comprising a polynucleotide according to any one of items 9 to 16, a cell or cell-free expression system according to item 17, or an oil decomposition agent according to item 18 or 19 is allowed to act on a subject to be treated.

(item 22)

The method according to item 21, wherein the treatment target comprises trans fatty acid or trans fatty acid-containing fat or oil.

(item 23)

A cleaning formulation, comprising: a polypeptide according to any one of items 1 to 8, a cell or cell-free expression system comprising a polynucleotide according to any one of items 9 to 16, or a cell or cell-free expression system according to item 17.

(item 24)

Use of the polypeptide of any one of items 1 to 8, or a cell or cell-free expression system comprising the polynucleotide of any one of items 9 to 16, the cell or cell-free expression system of item 17, or the oil decomposer of item 18 or 19 in a fat-modifying/oil-producing technology (transesterification or the like).

In the present disclosure, it is intended that 1 or more of the features described above can be provided not only in an explicit combination but also in another combination. Still further embodiments and advantages of the present disclosure will be appreciated to those of ordinary skill in the art upon reading and understand the following detailed description, as needed.

ADVANTAGEOUS EFFECTS OF INVENTION

By using the lipase of the present disclosure, it becomes easy to handle trans-fatty acid-containing oils and fats, which have been difficult to remove. Further, it is possible to remove fats and oils and to perform oil treatment in both high-temperature environments and low-temperature environments in which microorganisms and enzymes have not been used. Therefore, the novel lipase of the present disclosure is useful in the technical fields of detergents, leather industry, food industry, cleaning of environments contaminated with fats and oils, waste treatment such as garbage disposal, composting, and drainage treatment, chemicals such as composters and digestants, and cosmetics for oily skin.

Drawings

FIG. 1 shows the expression level of a gene encoding the lipase of the present disclosure when oils and fats are added. After the KH-1 strain was cultured in an inorganic salt medium containing triolein, oleic acid, elaidic acid or triolein at 28 ℃ for 6 hours, RNA was extracted from the KH-1 strain, and the expression level of the lipase gene of the present disclosure was quantified by quantitative RT-PCR. Ole indicates the results in oleic acid culture, TOle indicates the results in triolein culture, ED indicates the results in elaidic acid culture, and TED indicates the results in triolein culture. The expression level was expressed as a relative value normalized by the expression level of rpoD gene and assuming that the expression level in oleic acid culture or in triolein was 1. (A) The expression level under triolein was defined as 1, and (B) was defined as 1. The upper row represents the expression level of a representative sequence of the 1 st lipase gene, and the lower row represents the expression level of a representative sequence of the 2nd lipase gene. Error bars indicate standard deviation.

FIG. 2 shows the purification of the 1 st lipase of the present disclosure having a representative sequence from the culture supernatant of KH-1 strain obtained by culturing at 28 ℃. The culture supernatant of KH-1 strain obtained by culturing at 28 ℃ was purified by hydrophobic column chromatography, and the results of analyzing the eluted fractions by CBB staining were shown. The number of elution fractions is shown from the left, and the molecular weight is shown on the left.

FIG. 3 shows the purification of a 2nd lipase of the present disclosure having a representative sequence from the culture supernatant of KH-1 strain obtained by culturing at 15 ℃. The culture supernatant of KH-1 strain obtained by culturing at 15 ℃ was purified by hydrophobic column chromatography. Results of analysis of eluted fractions using CBB staining are shown. The number of elution fractions is shown from the left, and the molecular weight is shown on the left.

FIG. 4 shows, from the left side, the temperature optimum (A), thermostability (B), pH optimum (C) and pH stability (D) of the 1 st lipase having a representative sequence. The vertical axes of the graphs (A) to (D) indicate relative lipase activities, the horizontal axes of (A) and (B) indicate temperatures, and the horizontal axes of (C) and (D) indicate pH. (C) In the above-mentioned items (D), "good" means the result of the analysis using the acetic acid buffer at pH3.0 to 5.0, ". tangle-solidup" means the result of the analysis using the sodium phosphate buffer at pH5.0 to 7.0, "□" means the result of the analysis using the Tris-HCl buffer at pH7.0 to 9.0, and "Δ" means the result of the analysis using the CAPS buffer at pH9.0 to 11.0. Error bars indicate standard deviation.

FIG. 5 shows, from the left side, the optimum temperature (A), thermostability (B), optimum pH (C) and pH stability (D) of the 2nd lipase having a representative sequence. The vertical axes of the graphs (A) to (D) indicate relative lipase activities, the horizontal axes of (A) and (B) indicate temperatures, and the horizontal axes of (C) and (D) indicate pH. (C) In the above-mentioned items (D), "good" means the result of the analysis using the acetic acid buffer at pH3.0 to 5.0, ". tangle-solidup" means the result of the analysis using the sodium phosphate buffer at pH5.0 to 7.0, "□" means the result of the analysis using the Tris-HCl buffer at pH7.0 to 9.0, and "Δ" means the result of the analysis using the CAPS buffer at pH9.0 to 11.0. Error bars indicate standard deviation.

FIG. 6 shows the results of comparing the ventilator fouling decomposition activity of culture supernatants containing 1 st and 2nd lipases with that of oil detergents and general detergents. The upper left filter is a pre-treatment filter to which oil is adhered, and the center and right side of the upper row show filters immersed in KH-1 culture supernatant for 30 minutes and 1 hour, respectively. The center and right side of the middle show the filter impregnated with the oil wash for 2 and 4 hours, respectively. The center and right side of the lower row show the filters impregnated in the general cleaning agent for 2 hours and 4 hours, respectively.

FIG. 7 shows comparison of ventilator fouling-decomposing activities of the 1 st lipase and the 2nd lipase having representative sequences with Novozym (registered trademark) 51032 lipase (Novozymes). (A) The left side shows a photograph of a ventilator filter after 30 minutes of immersion in Novozym51032 lipase (N-51032), and the right side shows a photograph of a Novozym51032 lipase solution after immersion in a ventilator filter. (B) The left side shows a photograph of the ventilator filter after 30 minutes of immersion in the 1 st lipase of the representative sequence, and the right side shows a photograph of the 1 st lipase solution of the representative sequence after immersion in the ventilator filter. (C) The left side shows a photograph of the ventilator filter after 30 minutes of immersion in the representative sequence of 2nd lipase, and the right side shows a photograph of the representative sequence of 2nd lipase solution after immersion in the ventilator filter.

FIG. 8 shows the decomposition activity of lard and shortening with representative sequences of the 1 st lipase. The 1 st lipase having a representative sequence was purified from KH-1 strain cultured at 28 ℃ by hydrophobic column chromatography. (A) The lard after incubation with a buffer or a representative sequence of the 1 st lipase is shown, (B) the shortening after incubation with a buffer or a representative sequence of the 1 st lipase is shown, and (C) the results of the separation of the extracted oil by thin layer chromatography are shown.

FIG. 9 shows the decomposition activity of lard and shortening with representative sequences of the 2nd lipase. The 2nd lipase having a representative sequence was purified from KH-1 strain cultured at 15 ℃ by hydrophobic column chromatography. (A) The lard after incubation with a buffer or a 2nd lipase having a representative sequence is shown, (B) the shortening after incubation with a buffer or a 2nd lipase having a representative sequence is shown, and (C) the result of separating the extracted oil by thin layer chromatography is shown.

FIG. 10 shows a treatment test of trans-fatty acid-containing wastewater with KH-1 strain and BR3200(BioRemove3200, Novozymes). (A) The results of separating oil and fat in drainage water by thin layer chromatography after feeding drainage water containing trans fatty acid and culturing KH-1 strain and BR 320024 hours or 48 hours, and (B) the oil concentration in drainage water after culturing KH-1 strain and BR3200 hours.

FIG. 11 shows the decomposition of triolein and glycerol trioleate at 37 ℃ using a 1 st lipase of representative sequence. The left side shows the breakdown of triolein and the right side shows the breakdown of glycerol trioleate. TOle represents triolein and TED represents triolein. The upper side of each plate shows the signal for triglyceride (triolein or glycerol trioleate) and the lower side shows the signal for free fatty acids (oleic or elaidic acid). The results of the treatment with the enzyme-free buffer alone are also shown as a control.

FIG. 12 shows the decomposition of triolein and glycerol trioleate by using a 2nd lipase of representative sequence at 37 ℃. The left side shows the breakdown of triolein and the right side shows the breakdown of glycerol trioleate. TOle represents triolein and TED represents triolein. The upper side of each plate shows the signal for triglyceride and the lower side shows the signal for free fatty acids. The results of the treatment with the enzyme-free buffer alone are also shown as a control.

Fig. 13 shows the decomposition of triolein and glycerol trioleate at 15 ℃ using the lipase of the present disclosure. A1 st lipase having a representative sequence and a 2nd lipase having a representative sequence were purified from the culture supernatant of KH-1 strain using a butyl sepharose column, mixed with glycerol trioleate or glycerol trioleate, and treated at 15 ℃ for 24 hours. The left side shows the treatment of triolein and the right side shows the treatment of triolein. In each plate, the left side shows the treatment with a solution containing a 2nd lipase having a representative sequence, and the right side shows the treatment with a solution containing a 1 st lipase having a representative sequence. The upper side of each plate shows the signal for triglyceride (triolein or glycerol trioleate) and the lower side shows the signal for free fatty acids (oleic or elaidic acid).

Fig. 14 shows fat modification of triolein using 1 st and 2nd lipases of the present disclosure. Shows the results of analyzing the reaction product of the 1 st and 2nd lipases having representative sequences or a separate buffer mixed with methanol and triolein, and a methyl oleate standard by TLC analysis. From above, each signal corresponds to methyl oleate, triolein, oleic acid and diglyceride in that order.

Fig. 15 shows fat modification of trans fatty acids using the 1 st and 2nd lipases of the present disclosure. (A) The results of analyzing a product obtained by mixing representative sequences of 1 st and 2nd lipases or a buffer solution alone with methanol and palmitoleic Acid (Palmitelaidic Acid) and a methyl palmitoleate standard by TLC analysis are shown. From above, each signal corresponds to methyl-and-palmitoleate in turn. (B) Shows the results of analyzing a product of a reaction in which 1 st lipase and 2nd lipase having representative sequences or a buffer solution alone were mixed with methanol and trans-isooleic acid (vaccenic acid), and a trans-isooleic acid methyl ester standard by TLC analysis. From above, each signal corresponds to trans-isooleic acid methyl ester and trans-isooleic acid in that order.

FIG. 16 shows lipolytic activity of variants of the 1 st lipase of the invention. The left side shows the decomposition of triolein (TOle) and the right side shows the decomposition of Triolein (TED). In each photograph, the left lane shows the results of treatment with a buffer alone, and the right lane shows the results of treatment with a 1 st lipase variant. In each photograph, the upper arrow corresponds to undecomposed fat and oil, and the lower arrow corresponds to free fatty acid.

FIG. 17 shows lipolytic activity of variants of the 2nd lipase of the invention. The left side shows the decomposition of triolein (TOle) and the right side shows the decomposition of Triolein (TED). In each photograph, the left lane shows the results of treatment with a buffer alone, and the right lane shows the results of treatment with a 2nd lipase variant. In each photograph, the upper arrow corresponds to undecomposed fat and oil, and the lower arrow corresponds to free fatty acid.

Detailed Description

The best mode is shown below to explain the present invention. Throughout this specification, the singular form of expressions should be understood to include the plural form of concepts as well, unless otherwise specified. Thus, articles in the singular (e.g., "a," "an," "the," etc. in english) should be construed to include the plural as well as the plural unless specifically stated otherwise. In addition, terms used in the present specification should be understood to have meanings commonly used in the field unless otherwise specified. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

Definitions of terms specifically used in the present specification and/or basic technical contents are appropriately described below.

(definition of terms)

In the present specification, the term "lipase" is a kind of esterase, and refers to an enzyme that reversibly catalyzes a reaction of hydrolyzing a neutral fat (glyceride) into a fatty acid and glycerol. The lipase of the present disclosure is EC3.1.1.3 in the case of the enzyme number (EC number), and belongs to triglyceride lipase.

In the present specification, lipase activity can be determined as follows: an enzyme reaction was carried out using an ester of palmitic acid and 4-nitrophenol, i.e., 4-nitrophenylpalmitate (4-NPP), as a substrate, and the amount of p-nitrophenol produced by hydrolysis of the ester was determined by measuring the absorbance at 410 nm. First, 4-NPP (18.9mg) was added to 3% (v/v) TritonX-100 (12ml), and dissolved at 70 ℃ to prepare a substrate solution. 1mL of the substrate solution, 0.9mL of ion-exchanged water, and 1mL of 150mM GTA buffer (adjusted to pH7.0 by adding NaOH or HCl to 150mM 3, 3-dimethylglutaric acid, 150mM Tris, and 150mM 2-amino-2-methyl-1, 3-propanediol) were added to a cuvette, and incubated at 28 ℃ for 5 minutes. To this, 0.1mL of culture supernatant was added, and the value at 410nm was measured with stirring. Regarding the lipase activity, the activity was measured by defining the amount of enzyme producing 1. mu. mol of 4-nitrophenol as 1 unit (U), and the unit per 1mL of culture supernatant was calculated.

The decomposition/consumption ability of fats and oils and fatty acids can be measured by analyzing fats and oils remaining in the medium and free fatty acids produced by hydrolysis by thin layer chromatography. To show a specific quantitative procedure, first, an equal amount of chloroform was added to the culture supernatant to extract the oil and fat. For 5 μ l of the extract, the ratio by volume of 96:4: a developing solvent comprising chloroform, acetone and methanol at a ratio of 1 was developed on a silica gel-coated plate. The plates were treated with molybdic acid polyhydrate to develop the oil.

In the present specification, the "trans fatty acid-containing oil or fat" refers to an oil or fat containing trans fatty acids. "trans fatty acid" is used in the sense commonly used in the art to denote an unsaturated fatty acid having a trans-type double bond. Trans fatty acids naturally exist in trace amounts as conjugated linoleic acid and trans-vaccenic acid, but are produced in large amounts in the production of saturated fatty acids by hydrogenation of unsaturated fatty acids in the oil and fat industry, and are also included in foods such as margarine and shortening. The trans fatty acid includes elaidic acid, elapalmitoleic acid (palmitoleic acid), trans-vaccenic acid, etc., and as mentioned in the present specification, the kind of trans fatty acid is not particularly limited. The ratio of trans fatty acid present in the trans fatty acid-containing oil and fat is not particularly limited.

In the present specification, the term "ability to assimilate a trans-fatty acid-containing oil or fat" means: is used for promoting the assimilation activity of trans-fatty acid-containing oil and fat by microorganisms. In the present specification, the term "assimilating trans-fatty acid-containing oil and fat" is used in the meaning generally used in the art, and means that a microorganism ingests trans-fatty acid-containing oil and fat as a nutrient source such as a carbon source. "assimilation" includes, in addition to hydrolysis to glycerol and free fatty acids, conversion to a portion of other substances.

In the present specification, the "ability to decompose trans fatty acid-containing oils and fats" refers to an activity of hydrolyzing trans fatty acid-containing oils and fats into glycerin and free fatty acids.

In the present specification, the "ability to decompose fats and oils" (at each temperature) "can be measured as follows. That is, the lipase can be purified by the method described in the present specification, and the measurement can be carried out by mixing the lipase with p-nitrophenylpalmitate at a constant temperature to be a temperature to be measured, and measuring the absorbance at 410 nm. The substitution scheme is as follows: the measurement can be carried out by recovering the culture supernatant of the KH-1 strain or a derivative thereof by the method described in the present specification, mixing the culture supernatant or a crude purified lipase solution containing lipase with p-nitrophenylpalmitate at a constant temperature, and measuring the absorbance at 410 nm.

In the present specification, the "optimum temperature" for the capability of decomposing fats and oils is used in the meaning generally used in the art, and means a temperature at which the activity of decomposing fats and oils is a desired level or more (for example, a temperature range in which 80% or more of the activity of the enzyme at the highest level is maintained, and in another embodiment, the maximum level). Specifically, the peak value of the activity of the lipase 1 of the present disclosure for decomposing fats and oils is about 60 ℃, and the activity of 80% or more of the peak value is maintained in the range of about 45 to 70 ℃. The peak value of the activity of the 2nd lipase of the present disclosure for decomposing fats and oils is about 50 ℃, and 80% or more of the activity of the peak value is maintained in the range of about 35 to 55 ℃. In the context of the optimum temperature of the lipase of the present disclosure, the optimum temperature of the 1 st lipase of the present disclosure is 45 to 75 ℃, preferably 50 to 70 ℃, more preferably 55 to 65 ℃, even more preferably 58 to 63 ℃, and most preferably 60 ℃. The optimum temperature of the 2nd lipase of the present disclosure is 35 to 55 ℃, preferably 40 to 55 ℃, and more preferably 45 to 50 ℃. In another embodiment, the lipase of the present disclosure can be modified so as to change the optimum temperature, and the lower limit of the optimum temperature can be 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and the like (or can be changed by 1 ℃ at a time within a temperature range between these), and the upper limit can be 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃ (or can be changed by 1 ℃ at a time within a temperature range between these).

In the present specification, "thermostable" has the same meaning as terms such as "temperature stability", "thermostability", and the like, and is used in the meaning generally used in the art, meaning that an enzyme maintains activity at high temperatures. It is known that enzymes are generally inactivated at a temperature of about 50 ℃ due to a change in molecular structure or the like. In contrast, the relative enzyme activity of the 1 st lipase of the present disclosure, for example, when the enzyme activity after 30 minutes of treatment at 30 ℃ is taken as 100%, is: the lipase of the present disclosure can be said to have high thermostability because it is 100% or more even after 30 minutes of treatment at a temperature ranging from about 30 to about 85 ℃ and retains about 30% of the enzyme activity even after 30 minutes of treatment at 90 ℃. For the 2nd lipase of the present disclosure, for example, the relative enzyme activity when the enzyme activity after treatment at 70 ℃ for 30 minutes is set as 100% is: the temperature of the mixture is maintained at 90% or more even after the mixture is treated at about 15 to about 75 ℃ for 30 minutes, and at about 80 ℃ even after the mixture is treated at 80 ℃. In the present specification, "maintaining thermal stability" and the like are expressed similarly: the enzyme activity after 30 minutes of treatment at about 30 ℃ was 100%, and the relative enzyme activity was maintained at about 50% or more.

In the present specification, the "optimum pH" for the ability to decompose fats and oils is used in the meaning generally used in the art, and means a pH at which the activity to decompose fats and oils is at a certain level or higher (for example, a pH range in which 80% or higher of the activity of the enzyme at the highest level is maintained, and in another embodiment, the maximum level is sometimes used). The peak values of the lipid-degrading activities of the 1 st lipase and the 2nd lipase of the present disclosure are both about pH9, and 80% or more of the peak values of the activities are maintained in the range of about pH7.5 to pH 9.5. In the context of referring to the optimal pH of the 1 st lipase and the 2nd lipase, the optimal pH of the lipases of the present disclosure (both 1 st and 2 nd) is pH7.5 to pH9.5, preferably pH8.0 to pH9.3, more preferably pH9.

In the present specification, "Burkholderia forestea (Burkholderia arboris)" means a species belonging to Burkholderia forest (arboris) in terms of biological classification. The burkholderia plantarii strain KH-1 in the present specification has a gene encoding at least 2 lipases (i.e., a representative sequence of "1 st lipase" and a representative sequence of "2 nd lipase" of the present disclosure). The lipase of the present disclosure includes a lipase produced from KH-1 strain belonging to burkholderia plantarii species or a derivative strain thereof, but is not limited thereto.

In the present specification, the term "cell-free expression system" is used in the meaning generally used in the art, and means a system for producing a recombinant protein of interest in vitro using a transcription and translation mechanism of a biomolecule extracted from a cell. The cell-free expression system for producing the lipase of the present disclosure is not particularly limited, and a prokaryotic cell-free expression system such as escherichia coli can be used.

In the present specification, the term "oil-treated component" refers to a component contributing to assimilation and decomposition of fat and oil. Specifically, the oil-and-fat composition includes a component which promotes dispersion of oil-and-fat such as a biosurfactant, a component which decomposes oil into fatty acid and glycerin, a component which decomposes fatty acid, a component which decomposes glycerin, a component which adsorbs oil and removes it from an object to be treated, and the like. In one embodiment, the oil-treated component comprises a biosurfactant produced by KH-1 strain.

In the present specification, "oil-decomposing agent" means: a preparation capable of decomposing fats and oils, which comprises as an active ingredient a KH-1 strain of Burkholderia forestea of the present disclosure or a 1 st lipase or a 2nd lipase of the present disclosure produced by the strain. In the present disclosure, the oil-breaking agent may be used in combination with an oil-treating ingredient. In this case, the oil-disintegrating agent and the oil-treating component may be used together or may be used first. Further, the oil-decomposing agent may contain components (e.g., a carbon source and a nitrogen source) for improving the activity of the strain or lipase derived from the strain to be used, a surfactant, a drying protective agent, components for maintaining the bacteria for a long period of time, a preservative, an excipient, a reinforcing agent, an antioxidant, and the like.

The oil-disintegrating agent provided by the present disclosure is provided in a liquid or solid or dry state. Examples of the liquid form include a culture solution of bacteria (which may be concentrated or diluted as necessary), a culture supernatant, a form in which an enzyme component derived from the culture solution is adsorbed to a support, a form in which an enzyme is separated and purified from the culture solution and then suspended in a solvent, and the like. Examples of the solid include a form in which the solid is suspended in a solvent containing a protective agent such as glycerin and the like and frozen, a form in which the solid is fixed to a carrier (a form in which the solid is adsorbed to the carrier by a covalent bond, electrostatic interaction, hydrophobic interaction, or the like, a form in which the solid is molecularly crosslinked with the carrier, or the like), a form in which the solid is dehydrated by centrifugal separation, compression by a compressor, or the like, and a dried product obtained by drying. In a preferred embodiment, the oil-disintegrating agent can be provided typically in the form of a liquid, powder, or granule, and can be provided together with other components as a cleaning agent or a cleaning agent component.

Without intending to be limiting, as used herein, a "derivative," "analog," or "mutant" of a (lipase, etc.) preferably comprises a molecule that comprises a region that is substantially homologous to a subject protein (e.g., a lipase), such a molecule, in various embodiments, being at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to an aligned sequence between amino acid sequences of the same size, or when aligned using computer homology programs well known in the art, or a nucleic acid encoding such a molecule being capable of hybridizing to a sequence encoding a component protein under (high), moderate, or non-stringent conditions. This is a product obtained by modifying a protein by the substitution, deletion and addition of an amino acid, and this derivative is a protein which shows the biological function of the original protein, although not necessarily to the same extent. For example, the biological function of such proteins can be investigated by in vitro tests described in the present specification or known in the art and suitably utilized. As used herein, "functional activity" or "having functional activity" means in the present specification: the polypeptide of the present disclosure, i.e., the form related to the fragment or derivative, has a structural function, a control function, or a biochemical function of the protein such as biological activity.

In the present disclosure, a fragment of a lipase refers to a polypeptide including any region of a lipase, and may not necessarily have all the biological functions of a natural lipase as long as the object of the present disclosure (for example, degradation of trans-fatty acid-containing oils and fats) can be achieved.

In the present specification, "protein", "polypeptide", "oligopeptide" and "peptide" are used in the same sense in the present specification, and refer to an amino acid polymer of any length. The polymer may be linear or branched, or may be cyclic. The amino acid may be a natural or unnatural amino acid, or may be a modified amino acid. The term also includes those that assemble into a complex of multiple polypeptide chains. The term also includes natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., binding to a labeling component). This definition also includes, for example: polypeptides comprising 1 or more than 2 amino acid analogs (e.g., comprising unnatural amino acids, etc.), peptide-like compounds (e.g., peptidomimetics), and other modifications known in the art. In the present specification, "amino acid" is a general term for an organic compound having an amino group and a carboxyl group. When the protein or enzyme of the embodiments of the present disclosure includes a "specific amino acid sequence", any amino acid in the amino acid sequence may be chemically modified. In addition, any amino acid in the amino acid sequence may form a salt or a solvate. In addition, any amino acid in the amino acid sequence may be in L-type or D-type. In the above case, the protein according to the embodiment of the present disclosure may be said to include the above-mentioned "specific amino acid sequence". Examples of chemical modifications that amino acids contained in proteins undergo in vivo include N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, addition of glycosylphosphatidylinositol, etc.), side chain modifications (e.g., phosphorylation, addition of sugar chains, etc.), and the like. The amino acid may be a natural amino acid or an unnatural amino acid as long as the purpose of the present disclosure is satisfied.

In the present specification, "polynucleotide", "oligonucleotide" and "nucleic acid" are used in the same sense in the present specification, and refer to a nucleotide polymer of an arbitrary length. The term further includes "oligonucleotide derivatives" or "polynucleotide derivatives". "oligonucleotide derivative" or "polynucleotide derivative" includes derivatives of nucleotides, or refers to oligonucleotides or polynucleotides having unusual linkages between nucleotides, and may be used interchangeably. Specific examples of such oligonucleotides include 2 ' -O-methyl-ribonucleotides, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides have been changed to phosphorothioate bonds, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides have been changed to N3 ' -P5 ' phosphoramide bonds, oligonucleotide derivatives in which ribose and phosphodiester bonds in oligonucleotides have been changed to peptide nucleic acid bonds, oligonucleotide derivatives in which uracil in oligonucleotides has been replaced with C-5 propynyl uracil, oligonucleotide derivatives in which uracil in oligonucleotides has been replaced with C-5 thiazolyluracil, oligonucleotide derivatives in which cytosine in oligonucleotides has been replaced with C-5 propynyl cytosine, oligonucleotide derivatives in which cytosine in oligonucleotides has been replaced with phenoxazine-modified cytosine, and the like, An oligonucleotide derivative in which ribose in DNA is replaced with 2 '-O-propylribose, an oligonucleotide derivative in which ribose in an oligonucleotide is replaced with 2' -methoxyethoxyribose, and the like. Unless otherwise indicated, a particular nucleic acid sequence, as with the explicitly indicated sequences, includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions can be obtained by preparing sequences in which the 3 rd position of 1 or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-. In the present specification, "nucleic acid" can also be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. In the present specification, a "nucleotide" may be a natural nucleotide or a non-natural nucleotide.

In the present specification, "gene" refers to a factor that defines a genetic shape, and "gene" sometimes refers to "polynucleotide", "oligonucleotide", and "nucleic acid".

In the present specification, "homology" of a gene means the degree of identity between 2 or more gene sequences, and usually "homology" means a high degree of identity or similarity. Thus, the higher the homology of 2 genes, the higher their sequence identity or similarity. Whether or not 2 genes have homology can be determined by direct sequence comparison or hybridization under stringent conditions in the case of nucleic acids. In a direct comparison of 2 gene sequences, DNA sequences are homologous to each other when they are at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical, in the representative case between the gene sequences. Thus, in the present specification, "homolog" or "homologous gene product" refers to: proteins in other species, preferably microorganisms, more preferably bacteria, having the same biological functions as protein components in the complex described separately in the present specification. Such homologues are sometimes also referred to as "orthologous gene products". It is understood that such homologues, homologous gene products, orthologous gene products, and the like may also be used so long as they meet the objectives of the present disclosure.

In the present specification, an amino acid is represented by either of commonly known 3-letter symbols or one-letter symbols suggested by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are indicated by the commonly known single-letter symbols. In the present specification, BLAST, which is a tool for sequence analysis, is used for comparison of similarity, identity, and homology between amino acid sequences and nucleotide sequences, and default parameters (default parameters) are used for calculation. The identity search can be performed using, for example, BLAST2.7.1 (published by 2017.10.19) at NCBI. The value of "identity" in this specification generally means: values when aligned under default conditions using BLAST as described above. Among these, when a higher value is found by changing the parameter, the highest value is regarded as the homology value. When the identity is evaluated in a plurality of regions, the highest value among them is regarded as the homology value. "similarity" is a numerical value calculated in consideration of similar amino acids in addition to homology.

In one embodiment of the present disclosure, the "number" may be 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2, for example, and may be any one or less of the above values. Polypeptides which are known to be deleted, added, inserted or substituted with other amino Acids by 1 or several amino acid residues maintain their biological activity (Mark et al, Proc Natl Acad Sci USA.1984 Sep; 81(18): 5662-. The protein in which a deletion or the like has occurred can be produced by, for example, site-directed mutagenesis, random mutagenesis, biopanning (biopanning) using a protein phage library, or the like. As the site-directed Mutagenesis method, for example, KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) can be used. Selection of a protein having the same activity as that of the wild-type protein from among the variant proteins into which a deletion or the like has been introduced can be achieved by various identifications such as FACS analysis and ELISA.

In one embodiment of the present disclosure, "70% or more" as a numerical value such as homology may be, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%, and may be a range between any 2 values among the numerical values described above as starting points. The "homology" is obtained by calculating the ratio of the number of identical amino acids in 2 or more amino acid sequences according to the above-mentioned known method. Specifically, before calculating the ratio, the amino acid sequences in the amino acid sequence group to be compared are aligned, and a blank space is introduced into a part of the amino acid sequence when it is necessary to maximize the ratio of the same amino acid. Methods for alignment, methods for ratio calculation, methods for comparison, and computer programs related thereto are well known in the art (e.g., BLAST, etc., as described above). In the present specification, "identity" and "similarity" may be expressed by BLAST-determined values of NCBI, unless otherwise specified. In the algorithm for comparison of amino acid sequences by BLAST, Blastp may be used as a default. The measurement results were quantified in Positives or Identites format.

As used herein, the phrase "polynucleotide that hybridizes under stringent conditions" refers to conditions that are conventionally known in the art. Such a polynucleotide can be obtained by colony hybridization, plaque hybridization, southern blot hybridization, or the like, using a polynucleotide selected from the polynucleotides of the present disclosure as a probe. Specifically, the following nucleic acids are used: a polynucleotide can be identified when hybridization is performed at 65 ℃ in the presence of 0.7 to 1.0M NaCl using a filter to which DNA derived from a colony or a plaque is immobilized, and then the filter is washed at 65 ℃ with a 0.1 to 2-fold concentration of SSC (citrate buffer, saline-sodium citrate) solution (1-fold concentration of SSC solution having a composition of 150mM sodium chloride and 15mM sodium citrate). The "stringent conditions" may be, for example, the following conditions. (1) For washing, low ionic strength and high temperature (e.g., 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate at 50 ℃), (2) use of denaturants such as formamide in hybridization (e.g., 50% (v/v) formamide and 0.1% bovine serum albumin/0.1% polysaccharide (ficoll)/0.1% polyvinylpyrrolidone/50 mM ph6.5 sodium phosphate buffer, and 750mM sodium chloride, 75mM sodium citrate at 42 ℃), or (3) incubation of a low molecular weight SSC in a solution containing 20% formamide, 5 x SSC, 50mM sodium phosphate (ph7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20mg/ml denatured sheared salmon sperm DNA at 37 ℃, followed by washing the filter at about 37-50 ℃ with 1 x SSC. The formamide concentration may be 50% or more. The wash time may be 5, 15, 30, 60, or 120 minutes or more. As factors influencing the stringency of hybridization reactions, there are several factors such as temperature and salt concentration, and reference is made in detail to Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995). Examples of "high stringency conditions" are: 0.0015M sodium chloride, 0.0015M sodium citrate, 65-68 ℃, or 0.015M sodium chloride, 0.0015M sodium citrate and 50% formamide, 42 ℃. Hybridization can be carried out according to the methods described in the Protocols of Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, oxygen University Press (1995), and the like. Here, sequences containing only an a sequence or only a T sequence are preferably excluded from sequences that hybridize under stringent conditions. Moderately stringent conditions can be readily determined by, for example, one skilled in the art based on the length of the DNA, in the manner disclosed by Sambrook et al in Molecular Cloning: ALaborory Manual, phase 3, Vol.1, 7.42-7.45Cold Spring Harbor Laboratory Press,2001, and including, for nitrocellulose filters, hybridization conditions using 5 XSSC, 0.5% SDS, a prewash solution of 1.0mM EDTA (pH8.0), about 50% formamide at about 40-50 ℃,2 XSSC-6 XSSC (or other similar hybridization solutions such as Stark's solution in about 50% formamide at about 42 ℃), and wash conditions of 0.5 XSSC, 0.1% SDS at about 60 ℃. Thus, a polypeptide used in the present disclosure also includes a polypeptide encoded by a nucleic acid molecule that hybridizes under high or moderate stringency conditions with a nucleic acid molecule encoding a polypeptide as specifically described in the present disclosure.

The lipase of the present disclosure may preferably be a "purified" or "isolated" lipase. In the present specification, a "purified" substance or biological factor (e.g., nucleic acid or protein, etc.) refers to: a product from which at least a portion of a factor naturally associated with the substance or biological factor is removed. Thus, the purity of a biological factor in a purified biological factor is typically higher than the purity of the biological factor in the presence of the biological factor (i.e., is concentrated). The term "purified" as used in this specification means: preferably at least 75 wt.%, more preferably at least 85 wt.%, even more preferably at least 95 wt.%, and most preferably at least 98 wt.% of the biological factors of the same type are present. The substance or biological factor used in the present disclosure is preferably a "purified" substance. As used herein, an "isolated" substance or biological factor (e.g., nucleic acid or protein, etc.) refers to: substantially free of products of factors naturally associated with the substance or biological factor. The term "isolated" used in the present specification varies depending on its purpose, and thus sometimes does not need to be expressed in terms of purity, and means, when necessary: preferably at least 75 wt.%, more preferably at least 85 wt.%, even more preferably at least 95 wt.%, and most preferably at least 98 wt.% of the biological factors of the same type are present. The substance used in the present disclosure is preferably an "isolated" substance or biological factor.

As used herein, the term "fragment" or "fragment" is used in the same sense and refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n). The length of the fragment may be appropriately changed depending on the purpose, and for example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and 50 or more amino acids in the case of polypeptide, and the length (for example, 11) represented by an integer not listed herein is also suitable as the lower limit. In the case of polynucleotides, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and 100 or more nucleotides are exemplified, and lengths (for example, 11 and the like) represented by integers not enumerated herein are also suitable as lower limits. In the present specification, such fragments are understood as follows: for example, in the case where the fragment itself functions as a lipolytic molecule when the fragment is full-length, it is within the scope of the present disclosure that only the fragment itself functions as a lipolytic molecule.

In the present specification, the term "biological function" as used herein in reference to a gene or a nucleic acid molecule or polypeptide related thereto means a specific function that the gene, nucleic acid molecule or polypeptide may have in vivo or in vitro, and examples thereof include, but are not limited to, degradation of fats and oils (e.g., degradation of trans-fatty acid-containing fats and oils). In the present disclosure, for example, in addition to the trans-fatty acid-containing fat and oil, there may be mentioned a method of decomposing a cis-fatty acid-containing fat and oil, a method of decomposing a fat and oil containing a saturated fatty acid having no double bond, and the like, but the present invention is not limited thereto. In the present specification, a biological function can be exerted by a corresponding "biological activity". In the present specification, "biological activity" refers to an activity that a certain factor (for example, a polynucleotide, a protein, or the like) may have, and includes activities that exert various functions (for example, a degradation activity of trans-fatty acid-containing oils and fats). The "biological activity" may be an activity exerted in vivo or an activity exerted in vitro by secretion or the like. For example, where a factor is an enzyme, its biological activity includes its enzymatic activity. Such biological activity can be determined using techniques well known in the art. Thus, "active" means: indicates or indicates that binding (either direct or indirect) occurs; various measurable indicators that affect response (i.e., have a measurable effect in response to certain exposures or stimuli) may include, for example, affinity for a compound that binds directly to a polypeptide or polynucleotide of the disclosure, or a measure of the amount of protein upstream or downstream, e.g., after certain stimuli or events, or other similar functions.

In the present specification, "expression" of a gene, polynucleotide, polypeptide or the like means: the gene and the like are subjected to a certain action in vivo and become other forms. Preferably, the form of a polypeptide obtained by transcription and translation of a gene, a polynucleotide, or the like, and the form of mRNA formed by transcription are also an expression form. Thus, in the present specification, "expression product" includes such a polypeptide or protein, or mRNA. More preferably, the form of such a polypeptide may be one that has undergone post-translational processing. For example, the expression level of lipase can be determined by any method. Specifically, the expression level of lipase can be known by evaluating the amount of mRNA of lipase, the amount of lipase protein, and the biological activity of lipase protein. The amount of mRNA or protein of lipase can be determined by methods described in detail elsewhere in the specification or other methods known in the art.

In the present specification, "functional equivalents" mean: any substance having the same target function but a different structure from the target entity. Accordingly, functional equivalents of the "lipase" of the present disclosure are understood to include: a substance having a biological effect possessed by the lipase, which is not the lipase of the present disclosure itself, but is a mutant or variant thereof (e.g., amino acid sequence variant, etc.); and a substance which can be changed to a mutant or a variant having the biological effect of the lipase when acted upon (for example, a nucleic acid encoding the mutant or the variant, a vector or a cell comprising the nucleic acid). It is understood that in the present disclosure, functional equivalents of lipases can be used as well as lipases, even if not mentioned specifically. Functional equivalents may be found by searching a database or the like. In the present specification, "search" refers to a method of finding another nucleic acid base sequence having a specific function and/or property by using a certain nucleic acid base sequence electronically or biologically or by other methods. As electronic searches, BLAST (Altschul et al, J.mol. biol.215: 403-. Examples of the biological search include, but are not limited to, stringent hybridization, a microarray in which genomic DNA is placed on a nylon membrane or the like, a microarray in which genomic DNA is placed on a glass plate (microarray test), PCR, and in situ hybridization. In the present specification, it is intended that the gene used in the present disclosure includes a corresponding gene identified by such an electronic search or biological search.

As functional equivalents of the present disclosure, 1 or more amino acids may be inserted, substituted, or deleted in the amino acid sequence, or 1 or more amino acids may be added to one or both ends thereof. In the present specification, "1 or more amino acids are inserted, substituted or deleted in an amino acid sequence or 1 or more amino acids are added at one end or both ends thereof" means: modification is carried out by a known technique such as site-directed mutagenesis or substitution of a plurality of amino acids to such an extent that they can naturally occur by spontaneous mutation. The modified amino acid sequence may be, for example, an insertion, substitution, or deletion of 1 to 30, preferably 1 to 20, more preferably 1 to 9, still more preferably 1 to 5, and particularly preferably 1 to 2 amino acids, or an addition to one or both of the termini thereof. As for the modified amino acid sequence, the amino acid sequence thereof may preferably be an amino acid sequence having 1 or more (preferably 1 or more or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) conservative substitutions in the amino acid sequence of SEQ ID Nos. 4 to 6, 11 to 14, 16 or 18. Herein, "conservative substitution" refers to the substitution of 1 or more amino acid residues with other chemically similar amino acid residues in such a manner that the function of the protein is not substantially changed. Examples thereof include: substitution of a hydrophobic residue with another hydrophobic residue, substitution of a polar residue with another polar residue having the same charge, and the like. For functionally similar amino acids that can be subjected to such substitutions, each is well known in the art. Specific examples of the nonpolar (hydrophobic) amino acid include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Examples of the polar (neutral) amino acid include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of the (basic) amino acid having a positive charge include arginine, histidine, and lysine. Examples of the (acidic) amino acid having a negative charge include aspartic acid and glutamic acid.

In the present specification, "kit" means: typically divided into 2 or more blocks to provide units of the part(s) to be provided (e.g. enzymes, lipid breakdown agents, buffers, instructions, etc.). This form of kit is preferable when the object is to provide a composition, and the composition is a composition that should not be provided in a mixed state due to stability or the like, and is preferably used by mixing immediately before use. Advantageously, such a kit preferably has instructions or directions on how to use the provided moieties (e.g., enzymes, lipid-decomposing agents), or how to treat the reagents or the waste liquid after use. In the present specification, when the kit is used in the form of a reagent kit, the kit usually contains instructions describing the method of use of the enzyme, the fat-and-oil decomposer, and the like.

In the present specification, the "instruction" is a material for a user in which an instruction for a method of using the lipase of the present disclosure is described. The instruction book contains a text for instructing the method of using the lipase of the present disclosure. The instruction is prepared in accordance with a format prescribed by a regulatory agency of the country in which the present invention is implemented (for example, the ministry of health and labor, agriculture, forestry, aquatic products, etc. in japan, the Food and Drug Administration (FDA), the ministry of agriculture (USDA), etc. in the united states), and the subject of approval by the regulatory agency is clearly described. The instruction manual may be provided in the form of a paper medium, but is not limited thereto, and may be provided in the form of an electronic medium (e.g., a home page provided by the internet, an email) or the like, for example.

(preferred embodiment)

Preferred embodiments of the present disclosure are described below. The following embodiments are provided for better understanding of the present disclosure, and it is to be understood that the scope of the present disclosure should not be limited to the following descriptions. Therefore, it is obvious that those skilled in the art can appropriately make modifications within the scope of the present disclosure with reference to the description in the present specification. Further, it is to be understood that the following embodiments of the present disclosure may be used alone or in combination thereof.

(enzyme)

The present disclosure provides novel polypeptides.

As representative, the present disclosure provides a representative sequence of the 1 st lipase and a representative sequence of the 2nd lipase obtained from Burkholderia plantaris (Burkholderia arboris) KH-1 strain or a derivative thereof, and derivatives thereof.

In one embodiment, the polypeptide of the present disclosure may be:

(a) a polypeptide comprising the amino acid sequence shown in seq id No. 4, 11, 16 or 18;

(b) a polypeptide comprising an amino acid sequence comprising substitution, addition, deletion, or combination thereof of 1 or more amino acids in the amino acid sequence of (a) and having biological activity;

(c) a polypeptide having a sequence identity of at least 70% or more with the amino acid sequence represented by (a) or (b) and having a biological activity;

(d) a polypeptide comprising an amino acid sequence encoded by the nucleic acid sequence set forth in seq id No.1, 7, 15, or 17;

(e) a polypeptide having biological activity encoded by a nucleic acid sequence comprising substitution, addition, deletion or combination thereof of 1 or more nucleotides in the nucleic acid sequence shown in (d);

(f) a polypeptide encoded by a nucleic acid sequence having at least 70% or more sequence identity to the nucleic acid sequence of (d) or (e) and having biological activity;

(g) a polypeptide that is encoded by a nucleic acid sequence that hybridizes under stringent conditions to a polynucleotide that comprises a nucleic acid sequence represented by any one of (d) to (f) or a complementary sequence thereof, and that has biological activity;

(h) a polypeptide encoded by an allelic variant of any of the nucleic acid sequences of (d) to (g) and having biological activity; or

(i) A polypeptide comprising a fragment of the amino acid sequence shown in (a) to (h).

In another aspect, the present disclosure provides nucleic acid sequences encoding the novel polypeptides. The polynucleotide of the present disclosure may be:

(A) a polynucleotide comprising the nucleic acid sequence set forth in seq id No.1, 7, 15, or 17;

(B) a polynucleotide comprising a nucleic acid sequence comprising substitution, addition, deletion or combination thereof of 1 or more nucleotides in the nucleic acid sequence shown in (A);

(C) a polynucleotide comprising a nucleic acid sequence having at least 70% or more sequence identity to the nucleic acid sequence of (A) or (B) and encoding a polypeptide having biological activity;

(D) a polynucleotide encoding a polypeptide having a biological activity, the polynucleotide comprising a nucleic acid sequence that hybridizes under stringent conditions to a polynucleotide comprising the nucleic acid sequence shown in any one of (A) to (C) or a complementary sequence thereof;

(E) a polynucleotide encoding a polypeptide having a biological activity which is an allelic variant of any of the nucleic acid sequences (A) to (D); or

(F) A polynucleotide encoding a polypeptide comprising the amino acid sequence shown in seq id No. 4, 11, 16 or 18;

(G) a polynucleotide encoding a polypeptide having a biological activity, which comprises an amino acid sequence comprising substitution, addition, deletion or combination of 1 or more amino acids in the amino acid sequence of (F);

(H) a polynucleotide encoding a polypeptide having a sequence identity of at least 70% or more to the amino acid sequence represented by (F) or (G) and having a biological activity; or

(I) A polynucleotide comprising a fragment of the nucleic acid sequence shown in (F) to (H).

The biological activity of the polypeptide of the present disclosure may be at least one of any properties of the 1 st lipase or the 2nd lipase of the present disclosure, and may include, for example, an ability to assimilate a trans-fatty acid-containing oil or fat, an ability to decompose a trans-fatty acid-containing oil or fat, or both.

Thus, in one embodiment, the polypeptide encoded by the polypeptide or polynucleotide of the present disclosure may have an ability to assimilate trans-fatty acid-containing oils and fats, or an ability to decompose trans-fatty acid-containing oils and fats, or both.

In a preferred embodiment, the polypeptide encoded by the polypeptide or polynucleotide of the present disclosure has the ability to break down lipids at 15 ℃ or 10 ℃.

In another preferred embodiment, 45 to 75 ℃, 50 to 70 ℃, preferably 55 to 65 ℃, 58 to 63 ℃ or 60 ℃ is the optimum temperature for the ability to decompose oils and fats as the polypeptide encoded by the polypeptide or polynucleotide of the 1 st lipase of the present disclosure. The 2nd lipase polypeptide or the polypeptide encoded by the polynucleotide of the present disclosure is 35 to 55 ℃, 40 to 55 ℃, preferably 45 to 55 ℃, 45 to 50 ℃, or 50 ℃ which is the optimum temperature for its ability to decompose oils and fats.

In one embodiment, the polypeptide encoded by the polypeptide or polynucleotide of the 1 st lipase of the present disclosure may have thermostability at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, or 75 ℃ or higher, and the thermostability may be maintained at 50 ℃ or lower, 55 ℃ or lower, 60 ℃ or lower, 65 ℃ or lower, 70 ℃ or lower, 75 ℃ or lower, 80 ℃ or lower, 85 ℃ or lower, or 88 ℃ or lower. The polypeptide encoded by the polypeptide or polynucleotide of the 1 st lipase of the present disclosure preferably has a thermostability at 65 ℃ or higher, and can maintain the thermostability at 85 ℃ or lower. The polypeptide encoded by the polypeptide or polynucleotide of the 2nd lipase of the present disclosure may have thermal stability at 40 ℃ or higher, 45 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, or 75 ℃ or higher, and may retain the thermal stability at 40 ℃ or lower, 45 ℃ or lower, 50 ℃ or lower, 55 ℃ or lower, 60 ℃ or lower, 65 ℃ or lower, 70 ℃ or lower, 75 ℃ or lower, 80 ℃ or lower, or 85 ℃ or lower. The polypeptide encoded by the polypeptide or polynucleotide of the 2nd lipase of the present disclosure preferably has thermal stability at 50 ℃ or higher, and can maintain the thermal stability at 80 ℃ or lower.

In one embodiment, the polypeptide encoded by the polypeptide or polynucleotide of the present disclosure may be derived from Burkholderia plantarii (Burkholderia arboris), but is not limited thereto. In a preferred embodiment, it may be an enzyme derived from Burkholderia forestea KH-1 strain or a derivative strain thereof, but is not limited thereto.

In another aspect, the present disclosure provides an oil breakdown agent comprising: any of the polypeptides described above, or a cellular or cell-free expression system comprising the polynucleotide.

The cells of the disclosure comprise a 1 st lipase or 2nd lipase polypeptide of the disclosure, or incorporate a polynucleotide encoding a 1 st lipase or 2nd lipase of the disclosure in an expressible manner. The cell-free expression system provides a polynucleotide encoding the polypeptide of the 1 st lipase or 2nd lipase of the present disclosure in an expressible manner, and exerts an oil-decomposing effect by expressing the polypeptide by an appropriate mechanism.

In one embodiment, the oil breakdown agent comprises a further oil treatment component.

In another aspect, the present disclosure provides a kit for oil breakdown or oil treatment, the kit comprising a polypeptide of the present disclosure, or a cell or cell-free expression system of the present disclosure, or an oil breakdown agent; the kit is also provided with additional oil treatment ingredients. It will be understood that the oil-breaking and oil-treating ingredients contained in the kits of the present disclosure may be used in any combination with any of the species described elsewhere in this specification.

In another aspect, the present disclosure provides an oil breakdown removal method, including: the polypeptide of the present disclosure, or the cell or cell-free expression system of the present disclosure, or the oil-decomposing agent of the present disclosure is allowed to act on a treatment target. In the oil decomposition and removal method of the present disclosure, the treatment target preferably contains a trans-fatty acid-containing fat or oil, but is not limited thereto. Disclosed in this specification are: even when the subject does not contain trans-fatty acid-containing oils and fats, the lipase of the present disclosure can be used to highly efficiently decompose oils.

In another aspect, the present disclosure provides a cleaning agent comprising: a polypeptide of the present disclosure, a cell or cell-free expression system of the present disclosure, or an oil-splitting agent of the present disclosure.

In yet another aspect, there is provided a use in a fat modification/oil production technology (transesterification, etc.) using a polypeptide of the present disclosure, a cell or cell-free expression system of the present disclosure, or an oil-splitting agent of the present disclosure. As fat modification/oil production techniques (transesterification and the like), there can be mentioned: reaction to form ester compounds such as methyl ester and ethyl ester; displacement reaction of fatty acid contained in the oil; production of diglycerides, monoacylglycerols, and the like.

The polypeptides of the present disclosure are intended to include not only polypeptides having amino acid sequences of SEQ ID Nos. 4 to 6, 11 to 14, 16 or 18, but also variants thereof. Examples of such polypeptides include: a polypeptide having a biological activity, which comprises an amino acid sequence having substitution, addition, deletion or combination thereof of 1 or more amino acids with respect to the amino acid sequence of SEQ ID Nos. 4 to 6, 11 to 14, 16 or 18. In a specific embodiment, in the 1 st lipase representative sequence of the present invention, amino acids corresponding to the 1 st, 3 rd, 6 th, 137 th, 220 th, 227 th, 243 th, 276 th and 316 th positions in the amino acid sequence represented by SEQ ID NO. 4 are substituted. In another embodiment, in the 2nd lipase representative sequence of the present invention, amino acids at positions 13, 26, 45, 75, 100, 138, 168, 171, 214, 230, 234, 248, 250, 331 and 360 in the amino acid sequence shown in SEQ ID NO. 11 are substituted.

The present disclosure also provides nucleic acid sequences encoding the novel polypeptides, which are intended to include not only the nucleic acid sequences shown in SEQ ID Nos. 1 to 3, 7 to 10, 15 or 17, but also variants thereof. Examples of such a nucleic acid sequence include: a nucleic acid sequence comprising substitution, addition, deletion or combination thereof of 1 or more nucleotides relative to the nucleic acid sequence of SEQ ID NO.1 to 3, SEQ ID NO. 7 to 10, SEQ ID NO. 15 or SEQ ID NO. 17 and encoding a polypeptide having biological activity.

With respect to the positions in the above-mentioned amino acid sequences or nucleic acid sequences where variations can be introduced, those skilled in the art can easily determine the positions by homology search, motif search, domain analysis, alignment, secondary structure prediction, and the like. Regarding the position in the amino acid sequence or nucleic acid sequence at which a variation can be introduced, any position may be used as long as the variant having a variation at the position retains the lipase activity of the present disclosure, and variations may be introduced into a plurality of positions. Specifically, in the case of the 1 st lipase of the present disclosure, it is possible to have a mutation at any position of sequence No. 6 (full-length amino acid sequence), and for example, a mutation other than residues corresponding to S at position 131, D at position 308, and H at position 330 of sequence No. 6 is desirable, but the present invention is not limited thereto.

(production of enzyme)

As an example, the 1 st lipase or the 2nd lipase of the present disclosure may be purified from Burkholderia plantarii (Burkholderia arboris) KH-1 strain (deposit No. NITE BP-02731) or a derivative thereof. Specifically, the 1 st lipase of the present disclosure can be purified by a method comprising the following steps:

a step (a): culturing KH-1 strain or a derivative thereof in a medium comprising 1% canola oil at 28 ℃ for more than 24 hours;

a step (b): the culture supernatant of (a) was sterilized with a filter having a pore size of 0.45 μ M, and applied to a column containing about 10 times the column volume of 0.5M NaCl and 2mM CaCl₂Butyl-S Sepharose 6Fast Flow (GE Healthcare) equilibrated in 20mM Tris-HCl (pH7.0) buffer (hereinafter referred to as Tris buffer) was allowed to stand for about 1 hour to adsorb hydrophobic proteins to the column;

a step (c): washing the column of (b) with a Tris buffer solution in an amount of about 10 times the column volume, and then washing 5 times with a Tris buffer solution containing no NaCl in an amount of about 1 times the column volume, to elute proteins which are not strongly bound to the column carrier; and

step (d): the column of (c) was washed 4 times with a Tris buffer containing 0.5% Triton X-100 in an amount of about 1 times the column volume to elute proteins strongly bound to the column.

The 2nd lipase of the present disclosure can be purified by a method comprising the following steps:

a step (a): culturing KH-1 strain or a derivative thereof in a medium comprising 1% canola oil at 15 ℃ for more than 48 hours;

a step (b): the culture supernatant of (a) was sterilized with a filter having a pore size of 0.45 μ M, and applied to a column containing about 10 times the column volume of 0.5M NaCl and 2mM CaCl₂Butyl-S Sepharose 6Fast Flow (GE Healthcare) equilibrated in 20mM Tris-HCl (pH7.0) buffer (hereinafter referred to as Tris buffer) was allowed to stand for about 1 hour to adsorb hydrophobic proteins to the column;

a step (c): washing the column of (b) with a Tris buffer solution in an amount of about 10 times the column volume, and then washing 5 times with a Tris buffer solution containing no NaCl in an amount of about 1 times the column volume, to elute proteins which are not strongly bound to the column carrier; and

step (d): the column of (c) was washed 4 times with a Tris buffer containing 0.5% Triton X-100 in an amount of about 1 times the column volume to elute proteins strongly bound to the column.

In the method for purifying the enzyme, the specific conditions such as the culture conditions of the KH-1 strain or a derivative thereof and the conditions of column chromatography can be appropriately adjusted by those skilled in the art.

The 1 st lipase and the 2nd lipase of the present disclosure can also be produced by a secretory production system using a microorganism. Specifically, the 1 st or 2nd lipase of the present disclosure can be produced by a method comprising the following steps:

(a) a step of introducing a nucleic acid molecule encoding the mature form of the 1 st lipase or the 2nd lipase of the present disclosure (for example, SEQ ID NO: 1, 7, 15 or 17 or a modified sequence thereof) into pBIC1 plasmid (Takara Shuzo Co., Ltd.) and expressing the same in Bacillus brevis (Brevibacillus), and

(b) a step of purifying a protein secreted from Bacillus brevis (Brevibacillus) with Ni-agarose.

Alternatively, the recombinant lipase protein of the present disclosure can be produced by using expression systems such as the CORYNEX (registered trademark) system (monosodium glutamate) of Corynebacterium glutamicum (Corynebacterium glutamicum), the Pichia pastoris (ThermoFisher) expression system (Pichia pastoris), the baculovirus expression system (ThermoFisher) using insect cells, the protein secretion expression system (Customs) using Aspergillus, and the periplasmic secretion production system (Merck) of Escherichia coli using the pET system. In the method for producing lipase using a microorganism, specific conditions such as a method for expressing lipase, a host for expressing lipase, a method for purifying lipase secreted by a host, and the like can be appropriately adjusted by those skilled in the art. The lipase of the present disclosure can be expressed by various expression systems such as an in-vivo expression system and a cell-free expression system, in addition to a microbial secretion production system.

(use of enzyme)

The lipase of the present disclosure is useful in the treatment of fats and oils, and is used for the treatment of oil-and-fat-containing drainage, waste liquid, and the like. In this embodiment, the lipase of the present disclosure is used for wastewater treatment. When used for wastewater treatment, the lipase of the present disclosure can be used for industrial waste water, kitchen waste water, house waste water, and the like.

As a further example, the lipase of the present disclosure is used as a cleaning agent. When used as a detergent, the lipase of the present disclosure can be used in applications such as a detergent for washing, a detergent for kitchen, a detergent for cleaning, and a detergent for industrial use. The disclosed lipase-containing cleaners are particularly useful in drain cleaners such as pipe cleaners.

In another embodiment, the lipase of the present disclosure is used in fat modification/oil production technology. When used in fat-modifying/oil-and-fat-producing technologies, the lipase of the present disclosure can be used for food, industrial, fuel, and other applications.

In another embodiment, the lipase of the present disclosure is used as a countermeasure against environmental pollution. When used for environmental pollution countermeasures, the lipase of the present disclosure can be used for removing pollutants in soil pollution, groundwater pollution, marine pollution, and the like caused by oil.

In another embodiment, the lipase of the present disclosure is used for waste treatment and composting. When used for waste treatment and composting, the lipase of the present disclosure can be used for applications such as kitchen waste treatment including reduction, composting, feed-making of agricultural products and food wastes, pressurized floating separation devices, and reduction of oily sludge by a grease trap.

In another embodiment, the lipase of the present disclosure is used as a pharmaceutical product. The lipase of the present disclosure can be used as a digestant, a lipolysis promoter, or the like when used as a pharmaceutical.

In another embodiment, the lipase of the present disclosure is used as a cosmetic. The lipase of the present disclosure can be used for cosmetics for improving, preventing, or treating oily skin when used as cosmetics.

When the lipase of the present disclosure is used, it may be used in the form of a component containing the isolated enzyme, or in the form of a component containing the bacterium itself.

(general technique)

The Molecular biological, biochemical and microbiological methods used in this specification are well known and commonly used in the art, and include, for example, the methods described in Savli, H.Karadenizli, A.A., Kolayli, F.G., guides, S.A., Ozbek, U.S., Valiboglu, H.2003.expression stability of site for gene expression A promoter for gene expression standards of Pseudomonas aeruginosa, J.Med.Microbiol.52: 403. stone 408. Marie-Ange Teste, Man duquene 2004, Jean M and Jean-Luoic section 2009, simulation of Yeast for protein, Water drainage of biological, biological and biological methods of water use, such as "water drainage methods, biological methods of Water expression" 12, biological methods of Water drainage methods, biological methods of Water expression of microorganism, biological methods of Water expression products, biological methods of Water drainage methods of biological and environmental products of Water use, biological methods of biological and biological methods of Water drainage methods of Water expression products of biological and biological methods of Water production, biological and biological methods of Water drainage methods of Water production, biological and biological samples, biological methods of Water production, biological and biological methods of Water production, biological samples, biological and biological methods of Water production, biological methods of biological and biological samples, the relevant parts of these (which may be all) are incorporated herein by reference.

(remarks)

In the present specification, "or" is used when "at least 1 or more of the items listed in the text can be adopted. The same applies to "or". When it is explicitly stated in the present specification that "within a range of 2 values", the range also includes 2 values themselves.

All references cited in the present specification, such as scientific documents, patents, and patent applications, are incorporated by reference in their entirety into the present specification to the same extent as the contents of each of the individual references are specifically described.

The foregoing description shows preferred embodiments for ease of understanding the invention. The present invention will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is defined only by the claims.

Examples

The examples are described below. The biological treatments used in the following examples comply, where necessary, with the guidelines specified in the university of ancient houses, the regulatory bodies and the catahon protocol. Specifically, the reagents described in the examples were used, and they may be replaced with equivalents from other manufacturers (Sigma-Aldrich, Fuji film and Wako pure chemical industries, Nacalai Tesque, R & DSystems, USCN Life Science INC, Thermo Fisher Scientific, Kanto chemical, hunakoshi, Tokyo chemical, Merck, etc.).

Example 1 analysis of expression of putative Lipase

In this example, the expression analysis of the putative lipase gene found in KH-1 strain at each temperature is shown.

(Experimental method)

KH-1 strain was adjusted to OD at the final concentration based on the optical density of the cells obtained using HITACHI U-2810 spectrophotometer (Hitachi, Tokyo)₆₆₀Inoculating to canola oil-containing inorganic salt medium (Na) in a manner of 0.03₂HPO₄3.5g/L、KH₂PO₄ 2.0g/L、(NH₄)₂SO₄ 4.0g/L、MgCl₂·6H₂O 0.34g/L、FeSO₄·7H₂O 2.8mg/L、MnSO₄·5H₂O 2.4mg/L、CoCl₂·6H₂O 2.4mg/L、CaCl₂·2H₂O 1.7mg/L、CuCl₂·2H₂O 0.2mg/L、ZnSO₄·7H₂O0.3 mg/L, and NaMoO₄0.25mg/L) in 3L, and the culture was carried out at 28 ℃ or 15 ℃ in a 5L volume fermenter (250rpm, 200 ml/min under air reflux). Total RNA was extracted from each culture obtained by sampling with time using a High Pure RNA isolation kit (Roche). Using PrimeScript with 2. mu.g of total RNA as a template^TMThe RT reagent Kit with gDNA Eraser Perfect Real Time (Takara Bio Inc.) removed genomic DNA and synthesized cDNA. Thereafter, the cDNA stock solution was diluted 3-fold using a dilution solution attached to the kit. Using synthetic primers specific for the genes encoding the 1 st and 2nd lipases disclosed in the present disclosure, the primers were synthesized by Applied Biosystems StepOnePlus^TMQuantitative real-time RT-PCR was performed (Applied Biosystems). In the system containing PowerUp^TMPCR was carried out in 20. mu.l of a solution of SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific) (10. mu.l), each primer (final concentration: 0.5. mu.M), and cDNA (1. mu.l). The PCR reaction used a rapid cycling mode by performing 1 cycle at 95 deg.CAfter 2 minutes of denaturation, the procedure was repeated 40 times with a cycle of 3 seconds at 95 ℃ and 30 seconds at 60 ℃. Expression levels were normalized by the expression level of RNA polymerase sigma factor (rpoD). For the data, after confirming that the melting curve has a single peak, analysis was performed by a comparative Ct method (Δ Δ Ct method). The relative expression level of each lipase gene expressed in the LB medium as a control was 1.

(results)

The induction of expression of genes encoding 2 lipases (the 1 st lipase having a representative sequence and the 2nd lipase having a representative sequence) was confirmed by culturing KH-1 strain in the presence of an oil. As shown in Table 1 below, the gene encoding the 1 st lipase having a representative sequence showed a peak of expression level at about 24 hours when cultured at 28 ℃ and a peak of expression level at about 60 hours when cultured at 15 ℃. The gene encoding the 2nd lipase having a representative sequence showed a peak of expression level at about 24 hours when cultured at 28 ℃ and a peak of expression level at about 96 hours when cultured at 15 ℃.

[ Table 1]

TABLE 1 expression level of the gene encoding the lipase of the present invention in the canola oil-containing mineral salt medium (relative value when the expression level in LB medium is 1) at each culture temperature

	1 st Lipase	2nd Lipase
			28℃	136 +/-39 (24 hours)	198 +/-26 (24 hours)
15℃	57 +/-12 (60 hours)	134 + -28 (96 hours)

Cultivation time showing a peak for expression level in brackets.

Example 2 experiment of mutant

In this example, experiments using mutants of the lipase of the present disclosure are shown.

(Experimental method)

A polypeptide is produced by introducing mutations into the amino acid sequence (SEQ ID NO: 4) of the 1 st lipase of the present disclosure having representative sequences and the amino acid sequence (SEQ ID NO: 11) of the 2nd lipase having representative sequences, respectively, in such a manner that the sequence identity of amino acid residues other than the site known to be the active site in the lipase is 70 to 99%. The mutant polypeptide was used to measure the degradation activity of trans-fatty acid-containing oils and fats.

Example 3 Induction of expression of the 1 st and 2nd lipases of the present disclosure Using Trans fatty acids and Trans fatty acid-containing oils and fats)

In this example, the expression levels of the genes encoding the 1 st lipase and the 2nd lipase having representative sequences disclosed in the present disclosure were compared with those of cis fatty acid (oleic acid) and fatty acid containing cis fatty acid (triolein) when trans fatty acid (elaidic acid) and trans fatty acid-containing fat (triolein) were added.

(Experimental method)

KH-1 strain was cultured on a triolein plate for 2 days. After that, KH-1 strain was wiped with a cotton swab, suspended in PBS, and washed 2 times with PBS. As stock solution of oil and fat, TritonX-100 solution containing oleic acid, triolein, elaidic acid or triolein with final concentration of 2% and final concentration of 5% was prepared at 70 deg.CThe dissolution was carried out. The stock solutions of the respective oils and fats were added to 20ml of an inorganic salt medium at a volume ratio of 1/10, and washed KH-1 strain (oil and fat at a final concentration of 0.2%, Triton X-100 at a final concentration of 0.5%, OD of the strain) was inoculated thereto₆₆₀0.1). After 6 hours of incubation at 28 ℃, KH-1 strain was recovered, and total RNA was extracted from the recovered KH-1 strain. After cDNA was synthesized as described above using 2. mu.g of total RNA as a template, quantitative RT-PCR was performed using cDNA diluted 3-fold as a template. The expression level was normalized by the expression amount of rpoD gene, and compared with a relative value in which the expression level in oleic acid culture or in triolein was set to 1.

(results and investigation)

The expression level of the gene encoding the 1 st lipase having a representative sequence was increased by about 100-fold after the treatment with triolein (upper row of FIGS. 1A and 1B). This result indicates that trans fatty acid or trans fatty acid-containing oil and fat may be an inducer of the 1 st lipase. The expression of the gene encoding the 2nd lipase having a representative sequence was increased by about 10 to 20 times after the treatment with triolein (lower row in fig. 1A and 1B), and this result indicates that trans fatty acid or trans fatty acid-containing fat may also be an inducer of the 2nd lipase.

Example 4 purification of the 1 st Lipase from the supernatant obtained by culturing KH-1 Strain at 28 deg.C

In this example, it is shown that the 1 st lipase having a representative sequence was purified from the culture supernatant of KH-1 strain at 28 ℃.

(Experimental method)

4ml of Butyl-S Sepharose 6Fast Flow (GE Healthcare) were treated with a solution containing 0.5M NaCl and 2mM CaCl₂40ml of 20mM Tris-HCl (pH7.0) buffer was used for equilibration. The culture supernatant obtained by culturing the KH-1 strain at 28 ℃ was sterilized with a 0.45 μm filter and applied to a column. After allowing to stand for 1 hour to adsorb the hydrophobic protein, the mixture was washed with 40ml of the above buffer solution. Thereafter, the column carrier-weakly bound proteins were eluted by washing 5 times with 4ml of the aforementioned buffer solution containing no NaCl. Then, 4ml of the aforementioned buffer solution containing 0.5% Triton X-100 was added to wash 4 times to firmly form hydrophobic groups with the column carrierElution of water-bound proteins. Each eluted sample was filtered through a 0.45 μm filter, and to 20 μ l of the filtered sample, 4 μ l of 5 XSDS-PAGE sample buffer was added. After 5 minutes of heat treatment at 100 ℃ and centrifugation, 20. mu.l of the supernatant was applied to a polyacrylamide gel (5-12% gradient gel) and subjected to electrophoresis at 20mA for 90 minutes. Followed by CBB staining, whereby the proteins contained in the sample were subjected to separation analysis.

Analysis of the N-terminal amino acid sequence of the eluted lipase was performed. After concentrating the eluted fractions with a 30kDa cut-off spin column, the sample was applied to a surfactant removal column DetergentOut of spin column type^TM(TaKaRa), replaced by 2mM CaCl according to the manufacturer's protocol₂And 20mM Tris-HCl (pH7.0) to remove TritonX-100 used in elution. The obtained surfactant-removed lipase sample was separated by SDS-PAGE, and the N-terminal amino acid sequence was determined by Edman degradation after the target protein was blotted onto a PVDF membrane.

(results)

The protein purified from the culture supernatant at 28 ℃ showed a molecular weight of about 30kDa (FIG. 2). Furthermore, the lipase purified from the culture supernatant of KH-1 at 28 ℃ was almost a single band by hydrophobic column chromatography, indicating that it was almost composed of a single protein.

The N-terminal sequence of the protein purified from the culture supernatant at 28 ℃ was read and found to be Ala-Asp-Asn-Tyr-Ala, and it was confirmed that the protein was the product of the gene encoding the 1 st lipase.

(examination)

The amino acid sequence of the mature protein of the 1 st lipase having a representative sequence agrees with the sequence from the 45 th position of the product estimated from the gene sequence obtained from the genetic information of RAST, indicating that the amino acid residues at the 1 st to 44 th positions are a pro-sequence and are cleaved after endocrine secretion from the cell.

Example 5 purification of 2nd Lipase from supernatant obtained by culturing KH-1 Strain at 15 deg.C

In this example, it is shown that 2nd lipase having a representative sequence was purified from the culture supernatant of KH-1 strain at 15 ℃.

(Experimental method)

Lipase was purified from the culture supernatant obtained after the culture of KH-1 strain at 15 ℃ in the same manner as in example 4. The purified protein fractions were separated by SDS-PAGE, the target band was excised from the gel, decolorized and washed, and then Tris buffer (pH8.0) containing trypsin was added to the gel to carry out enzymatic digestion at 35 ℃ for 20 hours. The recovered sample solution was desalted and concentrated, and then analyzed by LC-MS/MS (Thermo Fisher Scientific Inc., USA).

(results)

The protein purified from the culture supernatant at 15 ℃ showed a molecular weight of about 40kDa (FIG. 3). Furthermore, the lipase purified from the culture supernatant of KH-1 at 15 ℃ was almost single-banded by hydrophobic column chromatography, indicating that it was almost composed of a single protein.

From the results of mass spectrometry of the protein purified from the culture supernatant at 15 ℃, an Asn-Val-Thr-Tyr-His fragment was detected, and a sequence at the N-terminal side thereof was not detected. Considering the specificity of the trypsin digestion, it is assumed that the product of the gene encoding the 2nd lipase has the N-terminal sequence of this sequence or Thr-Arg-Asn-Val-Thr-Tyr-His which is further extended by 2 residues to the N-terminal side.

(examination)

The amino acid sequence of the mature protein of the 2nd lipase having a representative sequence is identical to the sequence from position 49 or 51 of the product estimated from the gene sequence obtained from the genetic information of RAST, indicating that the amino acid residues at positions 1 to 48 or 50 are pro-sequences and are cleaved after endocrine in the cell.

(example 6 thermal stability, temperature optimum, pH optimum and pH stability of the Lipase of the present disclosure)

In this example, the thermostability, temperature optimum, pH optimum, and pH stability of the 1 st lipase and the 2nd lipase of the present disclosure are shown.

(Experimental method)

The 1 st lipase and the 2nd lipase having representative sequences of the present disclosure were purified from KH-1 strain cultured at 28 ℃ or 15 ℃ by hydrophobic column chromatography, respectively, to prepare 5U/ml. Each lipase activity was determined by measuring hydrolysis of p-nitrophenylpalmitate under the following conditions (A) to (D). (A) The 1 st lipase or 2nd lipase having a representative sequence of the present disclosure is mixed with a buffer adjusted to a temperature of 10 to 80 ℃, and the lipase activity is immediately measured at each temperature. (B) The 1 st or 2nd lipase of the present disclosure was incubated at a temperature between 30-95 ℃ for 30 minutes, after which the respective lipase activities were determined at 37 ℃. (C) The lipase activity was measured by mixing an acetate buffer (pH3.0 to 5.0), a sodium phosphate buffer (pH5.0 to 7.0), a Tris-HCl buffer (pH7.0 to 9.0) or a CAPS buffer (pH9.0 to 11.0) with the 1 st lipase or the 2nd lipase having a representative sequence of the present disclosure. The relative intensity was expressed by assuming that the lipase activity at pH9.0 was 100%. (D) Mixing each buffer used in (C) with a solution of 1 st lipase or 2nd lipase having a representative sequence of the present disclosure at a ratio of 1: 1, and left at 28 ℃ for 24 hours, after which the lipase activity was measured at pH 7.0.

(results)

The optimal temperature for the 1 st lipase of the present disclosure with a representative sequence was 60 ℃ and showed thermal stability over a wide temperature range between 30 ℃ and 85 ℃ (fig. 4). The optimum temperature for the representative sequence of the 2nd lipase of the present disclosure was 50 ℃ and thermal stability was shown in a wide temperature range between 15 ℃ and 80 ℃ (fig. 5). The optimal pH of the 1 st lipase of the disclosure with a representative sequence is pH9.0, and the stable pH is pH9.0-9.5. The most suitable pH of the 2nd lipase of the present disclosure having a representative sequence is pH9.0, and the stable pH is pH7.5 to 10.5.

(examination)

The results of the optimal temperature and thermal stability of the 1 st and 2nd lipases of the present disclosure indicate that the lipases of the present disclosure are lipases that maintain high activity also at high temperatures.

Example 7 comparison of ventilator fouling decomposition Activity of KH-1 Strain culture supernatant and detergent

In this example, the activities of decomposing dirt in a ventilation fan were compared between KH-1 strain culture supernatant and a detergent for oil and a general detergent.

(Experimental method)

As the culture supernatant of KH-1 strain, the supernatant of a culture obtained by culturing KH-1 strain in a medium containing 1% canola oil at 28 ℃ for 24 hours was used. As the oil detergent and the general detergent, a natural enzyme detergent Nicoeco kitchen (Nicoeco, long-field, 143-fold diluted with water according to the specification) and Family (registered trademark) (kao, tokyo, 666-fold diluted according to the specification) were used, respectively. The ventilator filter on which the oil stain was deposited was cut into 2cm square and placed in plates, and KH-1 strain culture supernatant (KH-1) and 5ml of a detergent for oil or a general detergent were added to each plate, followed by immersion for 30 minutes to 4 hours.

(results)

The results of example 7 are shown in FIG. 6. In general, detergent and oil detergent could not completely remove oil stains even in 4 hours of immersion washing, while culture supernatant of KH-1 strain was able to wash the filter to the same extent as a fresh product by 1 hour of immersion washing.

(examination)

Since it is considered that the lipase mainly contained in the culture supernatant obtained under the culture conditions is the enzyme of the present disclosure, the activity of decomposing the oil stains is considered to be brought about by the enzyme of the present disclosure.

Example 8 comparison of ventilator fouling decomposition Activity of KH-1 Strain-derived enzyme of the present disclosure with N-51032 enzyme

In this example, the ventilator soil-degrading activities of the 1 st and 2nd lipases of representative sequences of the present disclosure derived from strain KH-1 were compared with the Novozym51032 lipase (Novozymes).

(Experimental method)

The culture supernatant of KH-1 strain was purified by the above hydrophobic column chromatography, respectively, to obtain the 1 st lipase of the present disclosure having a representative sequence derived from KH-1 strain and the 2nd lipase having a representative sequence. Novozym51032 lipase (N-51032) was purchased from Novozymes corporation. Each lipase was dissolved in a solution containing 2mM CaCl₂And 0.25% TritonX-100 in 20mM Tris-HCl buffer (pH7.4) to a concentration of 15U/ml. Cutting the ventilator filter with deposited oil dirt into 2cm square and placing inTo each plate, 5ml of each lipase solution was added, and the plate was immersed for 30 minutes.

(results)

Fig. 7 shows the ventilator filter immersed in the lipase solution and the lipase solution after immersion. The scavenger fan filters impregnated with the 1 st and 2nd lipases of representative sequences of the present disclosure showed significant decomposition of oil stains compared to the scavenger fan filters impregnated with the Novozym51032 lipase.

Example 9 decomposition of lard and shortening with the presently disclosed No.1 Lipase

In this example, the decomposition activity of the 1 st lipase of the present disclosure having a representative sequence on lard and shortening is shown.

(Experimental method)

Using a catalyst containing 2mM CaCl₂And 0.5% TritonX-100 in 20mM Tris-HCl (pH7.0) elution buffer, and purifying representative sequence of the 1 st lipase of the present disclosure from the culture supernatant of KH-1 strain by hydrophobic column chromatography. 2ml of a solution of the lipase of the present disclosure (50U/ml) was added to the plate, to which 0.7g of lard (A) or 0.5g of shortening (B) was added. The solution is suitably mixed with stirring and left at 28 ℃ for 24 hours. The control was prepared by treating only the elution buffer described above.

(C) A stock solution was prepared by dissolving lard or shortening into 2.5% triton x-100 so that the final concentration reached 2%. 0.2ml of a stock solution in which lard and shortening were melted at 65 ℃ was added to 1.8ml of the above-mentioned lipase-containing buffer. The final concentrations of TritonX-100 and each oil were 0.25% and 0.2%, respectively. After 24 hours of incubation at 28 ℃ the oil was extracted with an equal amount of chloroform and 5. mu.l of the extract was subjected to thin layer chromatography.

(results)

The results of example 9 are shown in fig. 8. According to the experimental results of this example, lard and shortening treated with buffer alone were also in a solid state after treatment, while fats and oils treated with the 1 st lipase of representative sequence were dissolved ((a) and (B) of fig. 8). The results of thin layer chromatography also demonstrated that triglycerides in lard and shortening were decomposed into free fatty acids ((C) of fig. 8). It is apparent that the 1 st lipase of the present disclosure has the activity of hydrolyzing lard and shortening to make them soluble.

Example 10 decomposition of lard and shortening with the 2nd lipase of the present disclosure

In this example, the decomposition activity of the 2nd lipase of the present disclosure on lard and shortening is shown.

(Experimental method)

Using a catalyst containing 2mM CaCl₂And 0.5% Triton X-100 in 20mM Tris-HCl (pH7.0) elution buffer, and purifying the representative sequence of the 2nd lipase of the present disclosure by hydrophobic column chromatography from the culture supernatant of KH-1 strain cultured at 15 ℃ for 96 hours. 2ml of a solution of the lipase of the present disclosure (50U/ml) was added to the plate, to which 0.7g of lard (A) or 0.5g of shortening (B) was added. The solution was allowed to fuse with appropriate stirring and was left at 28 ℃ for 24 hours. The control was prepared by treating only the elution buffer described above.

(C) A stock solution was prepared by dissolving lard or shortening into 2.5% triton x-100 so that the final concentration reached 2%. 0.2ml of a stock solution in which lard and shortening were melted at 65 ℃ was added to 1.8ml of the above-mentioned lipase-containing buffer. The final concentrations of TritonX-100 and each oil were 0.25% and 0.2%, respectively. After 24 hours of incubation at 28 ℃ the oil was extracted with an equal amount of chloroform and 5. mu.l of the extract was subjected to thin layer chromatography.

(results)

The results of example 10 are shown in fig. 9. According to the experimental results of this example, lard and shortening treated with buffer alone were also in a solid state after treatment, while fats and oils treated with 2nd lipase of representative sequence were dissolved ((a) and (B) of fig. 9). It was also confirmed from the results of thin layer chromatography that triglyceride in lard and shortening was decomposed into free fatty acids ((C) of fig. 9). The 2nd lipase of the present disclosure, which showed representative sequence, had activity to hydrolyze lard and shortening, making them soluble.

Example 11 treatment test of Trans-fatty acid-containing wastewater with KH-1 Strain

In this example, a treatment test of trans-fatty acid-containing wastewater using lipase contained in KH-1 strain and BioRemove3200(BR3200, Novozymes) was shown.

(Experimental method)

(A) N (nitrogen) and P (phosphorus) were added to the drainage sample in amounts equivalent to the inorganic salt medium. KH-1 strain was cultured in LB medium, washed after the culture, and inoculated so that OD becomes 0.1, and subjected to lipase secretion. BR3200(BioRemove3200, Novozymes) was added in an amount of 10 times the concentration usually used. The drainage sample was incubated at 28 ℃ for 24 hours or 48 hours. Thereafter, the following solutions were used in a volume ratio of 96:4: a ratio of 1 contains developing solvents of chloroform, acetone and methanol, and the sample is developed on a silica gel-coated plate. The decomposition of the oil or fat was detected by color development using a polyhydracid molybdate using thin layer chromatography.

(B) For the drainage samples after 24 and 48 hours of treatment, the concentration of oil component corresponding to n-hexane extraction was measured by a reagent kit for measurement.

(results)

It was found that lipase secreted from KH-1 strain was able to decompose oil in the wastewater sample to about 50% of BR3200 in 24 hours of treatment (FIG. 10). Further, at 48 hours of treatment, the oil in the wastewater sample was decomposed to about 1/7 of BR3200 by the lipase secreted from KH-1 strain.

Example 12 decomposition of Each oil and fat with the 1 st Lipase of the present disclosure

In this example, the decomposition of triolein and glycerol trioleate by a 1 st lipase of the present disclosure having a representative sequence is shown.

(Experimental method)

As the 1 st lipase of the present disclosure having a representative sequence, a lipase purified by hydrophobic column chromatography from a culture supernatant obtained by culturing KH-1 strain in an inorganic salt medium containing 1% canola oil at 28 ℃ was used. Triolein and glycerol trioleate were dissolved in distilled water containing 5% of Triton X-100 and brought to a final concentration of 2% as stock solutions (10 × stock solutions). Will have representative sequence of the disclosure1 st Lipase solution (20 mM Tris buffer (containing 2mM CaCl) with a final concentration of 30U/ml₂And 0.5% TritonX-100; ph 7.0)) or the same buffer solution containing no lipase was dispensed into a tube in an amount of 100. mu.l each, and the above oil/fat 10 × stock solution was added at a volume ratio of 1/10. The final concentration of each oil is 0.2%, and the final concentration of TritonX-100 is 0.5%. These solutions were incubated at 37 ℃ for 24 hours. Thereafter, the following solutions were used in a volume ratio of 96:4: a ratio of 1 developing solvent comprising chloroform, acetone and methanol was developed on a silica gel-coated plate. The decomposition of the oil or fat was detected by color development using a polyhydracid molybdate using thin layer chromatography.

(results)

The 1 st lipase of the present disclosure having a representative sequence has an activity of hydrolyzing any of triolein and glycerol trioleate (fig. 11).

(examination)

It was clarified that the 1 st lipase of the present disclosure having a representative sequence has an activity of hydrolyzing both of triolein as a triglyceride of cis-form and triolein as a triglyceride of trans-form.

Example 13 degradation of Each oil and fat by the 2nd Lipase of the present disclosure

In this example, the decomposition of triolein and glycerol trioleate by a 2nd lipase of the present disclosure having a representative sequence is shown.

(Experimental method)

As the 2nd lipase of the present disclosure having a representative sequence, a lipase purified by hydrophobic column chromatography from a culture supernatant obtained by culturing KH-1 strain in an inorganic salt medium containing 1% canola oil at 15 ℃ was used. The 2nd lipase solution of the present disclosure (20 mM Tris buffer (containing 2mM CaCl) with a final concentration of 0.3U/ml₂And 0.5% TritonX-100; ph 7.0)) or the same buffer solution containing no lipase was dispensed into a tube in an amount of 100. mu.l each, and the above oil/fat 10 × stock solution was added at a volume ratio of 1/10. The final concentration of each oil is 0.2%, and the final concentration of TritonX-100 is 0.5%. These solutions were incubated at 37 ℃ for 22 hours. Thereafter, the following solutions were used in a volume ratio of 96:4: 1 ratio comprises chloroformAcetone and methanol on a silica gel coated plate. The decomposition of the oil or fat was detected by color development using a polyhydracid molybdate using thin layer chromatography.

(results)

The 2nd lipase of the present disclosure having a representative sequence has an activity of hydrolyzing both of triolein as a triglyceride of cis-form and triolein as a triglyceride of trans-form (fig. 12).

Example 14 degradation of Each oil and fat by the Lipase of the present disclosure at Low temperature

In this example, the decomposition of triolein and glycerol trioleate by 1 st and 2nd lipases with representative sequences of the present disclosure at 15 ℃ is shown.

(Experimental method)

The 1 st lipase having a representative sequence was purified by hydrophobic column chromatography from the culture supernatant obtained by culturing KH-1 strain in an inorganic salt medium containing 1% canola oil at 28 ℃. As the 2nd lipase having a representative sequence, a lipase purified by hydrophobic column chromatography from a culture supernatant obtained by culturing KH-1 strain at 15 ℃ in the same manner was used. For triolein or glycerol trioleate, the above 10 × stock solution was added at 1/20 to a final concentration of 0.1% to mix with the 1 st lipase or 2nd lipase solution. The 1 st lipase having a representative sequence was treated with 4-NPP as a substrate and the final concentration was set to 6U/ml, and the 2nd lipase having a representative sequence was treated with 4-NPP as a substrate and the final concentration was set to 6U/ml. These solutions were incubated at 15 ℃ for 72 hours. Thereafter, the following solutions were used in a volume ratio of 96:4: a ratio of 1 developing solvent comprising chloroform, acetone and methanol was developed on a silica gel-coated plate. The decomposition of the oil or fat was detected by color development using a polyhydracid molybdate using thin layer chromatography.

(results and investigation)

Triolein, which is a triglyceride of cis-form, is decomposed into oleic acid by treatment with either of 1 st lipase and 2nd lipase having representative sequences. Glycerol trioleate, which is a trans-form triglyceride, is also decomposed into elaidic acid by treatment with either of the 1 st lipase and the 2nd lipase (fig. 13). These results demonstrate that the 1 st lipase and the 2nd lipase having representative sequences according to the present disclosure have an activity of degrading either of a cis-fatty acid-containing fat and an trans-fatty acid-containing fat even under low temperature (15 ℃).

Example 15 modification of triolein fat and production of fatty acid methyl ester oil and fat

In this example, the fat modification of cis-form fatty acids by the 1 st lipase and 2nd lipase having representative sequences of the present invention is shown.

(Experimental method)

KH-1 strain was subjected to 48-hour fermentor culture in BS culture containing 1% canola oil, and 1 st and 2nd lipases with representative sequences were purified from their supernatants using a butyl sepharose column. The 1 st lipase and 2nd lipase solutions were adjusted to 5U/ml. Triolein was dissolved in methanol to reach 0.5% as a stock solution. A mixture of 80. mu.l of the stock solution and 20. mu.l of each enzyme solution was dispensed into a screw-cap tube, and the tube was left to stand in an incubator at 37 ℃ for 96 hours. In addition, a mixture with a buffer solution containing no lipase was used as a control zone. Then, 100. mu.l of water and 50. mu.l of chloroform were added to each solution, and after stirring sufficiently with a vortex mixer, the mixture was centrifuged at 12,000g for 5 minutes. For the lower chloroform layer of 10. mu.l, a solution of 80: 20: the developing solvent containing hexane, diethyl ether and acetic acid in a ratio of 1 was subjected to TLC analysis.

(results)

The results of example 15 are shown in fig. 14. Both of the 1 st lipase and the 2nd lipase of the present invention catalyze a transesterification reaction of triolein, which is triglyceride in a cis-form, to produce methyl oleate.

Example 16 fat modification of Trans fatty acid and production of fatty acid methyl ester oil

In this example, the fat modification of trans fatty acids by the 1 st lipase and the 2nd lipase having representative sequences of the present invention is shown.

(Experimental method)

KH-1 strain was subjected to 48-hour fermentor culture in BS culture containing 1% canola oil, and 1 st and 2nd lipases having representative sequences were purified from the supernatant using a butyl sepharose column. The 1 st lipase and 2nd lipase solutions were adjusted to 5U/ml. Each trans fatty acid monomer of (a) elaiopalmitoleic acid and (B) trans isooleic acid was dissolved in methanol so that each fatty acid monomer was 0.5%, and the resulting solution was used as a stock solution. A mixture of 80. mu.l of each trans-fatty acid stock solution and 20. mu.l of each enzyme solution was dispensed into a screw-cap tube, and the tube was allowed to stand in an incubator at 37 ℃ for 72 hours. In addition, a mixture with a buffer solution containing no lipase was used as a control zone. Then, 100. mu.l of water and 50. mu.l of chloroform were added to each solution, and after stirring sufficiently with a vortex mixer, centrifugation was performed at 12,000g for 5 minutes. For the lower chloroform layer of 5. mu.l, the following solutions were used in a volume ratio of 80: 20: the developing solvent containing hexane, diethyl ether and acetic acid in a ratio of 1 was subjected to TLC analysis.

(results)

The results of example 16 are shown in FIG. 15. Both of the 1 st lipase and the 2nd lipase of the present invention catalyze transesterification of tripalmitoleic acid and transisooleic acid, which are trans fatty acids, to produce methyl tripalmitoleate and methyl transisooleate, respectively. It was thus clear that these enzymes are also useful in the reaction of trans fatty acids other than elaidic acid.

Example 17 lipolytic Activity of the 1 st lipase variant of the present invention

In this example, the variant of the 1 st lipase of the present invention was analyzed for lipolytic activity.

(Experimental method)

An expression construct of a variant (SEQ ID NO: 16) obtained by adding the 1 st, 3 rd, 6 th, 137 th, 220 th, 227 th, etc. of the amino acid sequence of the 1 st lipase (SEQ ID NO: 4) having a representative sequence of the present invention to the amino acid sequence of Bacillus brevis expression system (BIC system: TaKaRa),The amino acids at positions 243, 276 and 316 are substituted with serine, aspartic acid, threonine, serine, valine, threonine, leucine, glutamine and lysine, respectively. The cells were cultured at 30 ℃ for 48 hours in a medium prepared by adding neomycin to 2SY medium to a final concentration of 50. mu.g/ml. 50ml of the supernatant of the cultured strain was applied to a butyl agarose column and eluted to 20mM Tris-HCl, 2mM CaCl containing 0.25% Triton X-100₂In (1). The recombinant lipase was added with AD +6xHis + DDDK (enterokinase recognition sequence) at the N-terminus of the mature sequence. As the oils and fats, the decomposition activity of each oil and fat was analyzed by TLC using glycerol trioleate (TOle) and glycerol Trioleate (TED) under the following experimental conditions:

kind of oil and fat: triolein, Trielaidic acid glyceride

Reaction solution: 20mM Tris-HCl (pH7.0),2mM CaCl₂,0.5％Triton X100

Final oil concentration: 0.2 percent of

The processing method: mu.l of the enzyme solution was added to a microcentrifuge tube (Eppendorf Tubes) and 1/5 amounts of 10 Xeach of the lipid stocks were added.

Treatment temperature: 37 deg.C

Processing time: 48 hours

Oil extraction: extraction was performed with half the amount of chloroform, and 10. mu.l was loaded for TLC.

Unfolding plates: silica gel coating plate

Developing solvent: chloroform: acetone: methanol 96:4:2

Detection: color development Using molybdate polyhydrate (2.4g/60ml EtOH)

(results)

The results of example 17 are shown in fig. 16. The variant having the above-mentioned variation at 9 also has high lipolytic activity.

Example 18 lipolytic Activity of the 2nd lipase variant of the present invention

In this example, the 2nd lipase variant of the present invention was analyzed for lipolytic activity.

(Experimental method)

An expression construct of a variant (SEQ ID NO: 18) in which the amino acids at positions 13, 26, 45, 75, 100, 138, 168, 171, 214, 230, 234, 248, 250, 331 and 360 of the amino acid sequence of the 2nd lipase (SEQ ID NO: 11) having a representative sequence of the present invention are substituted with leucine, arginine, isoleucine, valine, serine, glutamic acid, arginine, glycine, asparagine, serine, arginine, glycine, asparagine and alanine, was constructed according to an expression system (BIC system: TaKaRa) using a Bacillus brevis expression system. Adding MgCl with a final concentration of 20mM to the TM medium₂And neomycin at a final concentration of 50. mu.g/ml, at 30 ℃ for 72 hours. 50ml of the supernatant of the cultured strain was applied to a butyl agarose column and eluted to 20mM Tris-HCl and 2mM CaCl each containing 0.25% Triton X-100₂In (1). The recombinant lipase was added with AD +6xHis + DDDK (enterokinase recognition sequence) at the N-terminus of the predicted signal sequence-cleaved sequence. As the oils and fats, the decomposition activity of each oil and fat was analyzed by TLC using glycerol trioleate (TOle) and glycerol Trioleate (TED) under the following experimental conditions:

kind of oil and fat: triolein, Trielaidic acid glyceride

Reaction solution: 20mM Tris-HCl (pH7.0),2mM CaCl₂,0.5％Triton X100

Final oil concentration: 0.2 percent of

The processing method: mu.l of enzyme solution was added to the microfuge tube and 1/5 volumes of 10X each oil stock were added.

Treatment temperature: 37 deg.C

Processing time: 48 hours

Oil extraction: the mixture was extracted with an equal amount of chloroform, and 10. mu.l of the extract was applied for TLC.

Unfolding plates: silica gel coating plate

Developing solvent: chloroform: acetone: methanol 96:4:2

Detection: color development Using molybdate polyhydrate (2.4g/60ml EtOH)

(results)

The results of example 18 are shown in fig. 17. The variant having the above 15 mutations also has high lipolytic activity.

(remarks)

As above, the present invention has been illustrated using the preferred embodiments of the present disclosure, but it is understood that the present invention is to be construed only by the scope of the claims. It is to be understood that the contents of patents, patent applications, and other documents cited in this specification are incorporated by reference into this specification as if fully set forth herein. The present application claims priority to Japanese patent application 2018-129504, filed in the office on 7/6/2018, the contents of which are to be understood as being incorporated herein by reference.

Industrial applicability

The present disclosure is useful for treatment of trans-fatty acid-containing wastewater that is problematic in food factories and the like in decomposing trans-fatty acid-containing fats and oils.

Deposit number

NITE BP-02731

SEQ ID NO.1 mature sequence of nucleic acid sequence representative of Lipase 1 of the present disclosure

SEQ ID NO. 2 nucleic acid sequence including nucleotide encoding pro-sequence in nucleic acid sequence representative of the 1 st lipase of the present disclosure

SEQ ID NO. 3 full Length sequence of nucleic acid sequence representative of Lipase 1 of the present disclosure

SEQ ID NO. 4 mature sequence of amino acid sequence representative of the 1 st lipase of the present disclosure

SEQ ID NO. 5 amino acid sequence including pre-sequence among representative amino acid sequences of the 1 st lipase of the present disclosure

SEQ ID NO. 6 full Length sequence of amino acid sequence representative of Lipase No.1 of the present disclosure

SEQ ID NO. 7 mature sequence of nucleic acid sequence representative of Lipase 2 of the present disclosure

SEQ ID NO. 8 nucleic acid sequence including nucleotides encoding short-chain pro-sequence in nucleic acid sequence representative of lipase 2 of the present disclosure

SEQ ID NO. 9 nucleic acid sequence including nucleotide encoding long-chain pre-sequence in nucleic acid sequence representative of lipase 2 of the present disclosure

SEQ ID NO. 10 full Length sequence of nucleic acid sequence representative of 2nd Lipase of the present disclosure

SEQ ID NO. 11 mature sequence of amino acid sequence representative of 2nd lipase of the present disclosure

SEQ ID NO.12 amino acid sequence including short-chain pro-sequence among the 2nd lipase representative amino acid sequences of the present disclosure

SEQ ID NO. 13 amino acid sequence including long-chain pre-sequence among the representative amino acid sequences of the 2nd lipase of the present disclosure

SEQ ID NO. 14 full Length sequence of amino acid sequence representative of Lipase 2 of the present disclosure

SEQ ID NO. 15 engineered sequence of mature sequence of nucleic acid sequence representative of Lipase 1 of the present disclosure

SEQ ID NO. 16 engineered sequence of mature sequence of representative amino acid sequence of 1 st lipase of the present disclosure

SEQ ID NO. 17 engineered sequence of mature sequence of nucleic acid sequence representative of Lipase 2 of the present disclosure

SEQ ID NO. 18 modified sequence of mature sequence of representative amino acid sequence of 2nd lipase of the present disclosure

Sequence listing

<110> NATIONAL UNIVERSITY corporate famous ancient House UNIVERSITY (NATIONAL UNIVERSITY CORPORATION NAGOYA UNIVERSITY)

<120> novel lipase for decomposing trans-fatty acid-containing oils and fats

<130> NU002PCT

<150> JP 2018-129504

<151> 2018-07-06

<160> 18

<170> PatentIn version 3.5

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aaccaggatg cgcttgcatc gctgaagacg ctgacgaccg ctcaggcggc gacctataac 660

cagaactatc cgagcgcggg ccttggtgca ccgggcagct gccagaccgg cgcaccgacg 720

gaaacggtcg gcggcaacac gcatctgctg tattcgtggg ccggcacggc gatccagccg 780

acgctttcgc tgttcggcgt gacgggtgcg aaggacacga gcacgattcc gctcgtcgat 840

cccgcaaacg cgctcgaccc gtcgacgctc gcgctgttcg gcaccggcac ggtgatgatc 900

aaccgtggct cgggtcagaa cgacgggctc gtgtcgaagt gcagcgcgct gtacggcaag 960

gtgctgagca cgagctacaa gtggaaccac atcgacgaga tcaaccagct gctcggcgtg 1020

cgcggcgcgt atgcggaaga tccggtcgcg gtgatccgca cgcatgcgaa ccggctgcag 1080

ctcgcgggcg tgtaa 1095

<210> 4

<211> 320

<212> PRT

<213> Burkholderia foresta (Burkholderia arboris)

<400> 4

Ala Asp Asn Tyr Ala Ala Thr Arg Tyr Pro Ile Ile Leu Val His Gly

1 5 10 15

Leu Thr Gly Thr Asp Lys Tyr Ala Gly Val Leu Glu Tyr Trp Tyr Gly

20 25 30

Ile Gln Glu Asp Leu Gln Gln His Gly Ala Thr Val Tyr Val Ala Asn

35 40 45

Leu Ser Gly Phe Gln Ser Asp Asp Gly Pro Asn Gly Arg Gly Glu Gln

50 55 60

Leu Leu Ala Tyr Val Lys Thr Val Leu Ala Ala Thr Gly Ala Thr Lys

65 70 75 80

Val Asn Leu Val Gly His Ser Gln Gly Gly Leu Thr Ser Arg Tyr Val

85 90 95

Ala Ala Val Ala Pro Asp Leu Val Ala Ser Val Thr Thr Ile Gly Thr

100 105 110

Pro His Arg Gly Ser Glu Phe Ala Asp Phe Val Gln Ser Val Leu Ala

115 120 125

Tyr Asp Pro Thr Gly Leu Ser Ser Thr Val Ile Ala Ala Phe Val Asn

130 135 140

Val Phe Gly Ile Leu Thr Ser Ser Ser His Asn Thr Asn Gln Asp Ala

145 150 155 160

Leu Ala Ser Leu Lys Thr Leu Thr Thr Ala Gln Ala Ala Thr Tyr Asn

165 170 175

Gln Asn Tyr Pro Ser Ala Gly Leu Gly Ala Pro Gly Ser Cys Gln Thr

180 185 190

Gly Ala Pro Thr Glu Thr Val Gly Gly Asn Thr His Leu Leu Tyr Ser

195 200 205

Trp Ala Gly Thr Ala Ile Gln Pro Thr Leu Ser Leu Phe Gly Val Thr

210 215 220

Gly Ala Lys Asp Thr Ser Thr Ile Pro Leu Val Asp Pro Ala Asn Ala

225 230 235 240

Leu Asp Pro Ser Thr Leu Ala Leu Phe Gly Thr Gly Thr Val Met Ile

245 250 255

Asn Arg Gly Ser Gly Gln Asn Asp Gly Leu Val Ser Lys Cys Ser Ala

260 265 270

Leu Tyr Gly Lys Val Leu Ser Thr Ser Tyr Lys Trp Asn His Ile Asp

275 280 285

Glu Ile Asn Gln Leu Leu Gly Val Arg Gly Ala Tyr Ala Glu Asp Pro

290 295 300

Val Ala Val Ile Arg Thr His Ala Asn Arg Leu Gln Leu Ala Gly Val

305 310 315 320

<210> 5

<211> 351

<212> PRT

<213> Burkholderia foresta (Burkholderia arboris)

<400> 5

Val Ala Cys Ala Met Ser Val Ala Pro Phe Ala Gly Thr Thr Ala Leu

1 5 10 15

Met Thr Leu Ala Thr Thr His Ala Ala Met Ala Ala Thr Ala Pro Ala

20 25 30

Asp Asn Tyr Ala Ala Thr Arg Tyr Pro Ile Ile Leu Val His Gly Leu

35 40 45

Thr Gly Thr Asp Lys Tyr Ala Gly Val Leu Glu Tyr Trp Tyr Gly Ile

50 55 60

Gln Glu Asp Leu Gln Gln His Gly Ala Thr Val Tyr Val Ala Asn Leu

65 70 75 80

Ser Gly Phe Gln Ser Asp Asp Gly Pro Asn Gly Arg Gly Glu Gln Leu

85 90 95

Leu Ala Tyr Val Lys Thr Val Leu Ala Ala Thr Gly Ala Thr Lys Val

100 105 110

Asn Leu Val Gly His Ser Gln Gly Gly Leu Thr Ser Arg Tyr Val Ala

115 120 125

Ala Val Ala Pro Asp Leu Val Ala Ser Val Thr Thr Ile Gly Thr Pro

130 135 140

His Arg Gly Ser Glu Phe Ala Asp Phe Val Gln Ser Val Leu Ala Tyr

145 150 155 160

Asp Pro Thr Gly Leu Ser Ser Thr Val Ile Ala Ala Phe Val Asn Val

165 170 175

Phe Gly Ile Leu Thr Ser Ser Ser His Asn Thr Asn Gln Asp Ala Leu

180 185 190

Ala Ser Leu Lys Thr Leu Thr Thr Ala Gln Ala Ala Thr Tyr Asn Gln

195 200 205

Asn Tyr Pro Ser Ala Gly Leu Gly Ala Pro Gly Ser Cys Gln Thr Gly

210 215 220

Ala Pro Thr Glu Thr Val Gly Gly Asn Thr His Leu Leu Tyr Ser Trp

225 230 235 240

Ala Gly Thr Ala Ile Gln Pro Thr Leu Ser Leu Phe Gly Val Thr Gly

245 250 255

Ala Lys Asp Thr Ser Thr Ile Pro Leu Val Asp Pro Ala Asn Ala Leu

260 265 270

Asp Pro Ser Thr Leu Ala Leu Phe Gly Thr Gly Thr Val Met Ile Asn

275 280 285

Arg Gly Ser Gly Gln Asn Asp Gly Leu Val Ser Lys Cys Ser Ala Leu

290 295 300

Tyr Gly Lys Val Leu Ser Thr Ser Tyr Lys Trp Asn His Ile Asp Glu

305 310 315 320

Ile Asn Gln Leu Leu Gly Val Arg Gly Ala Tyr Ala Glu Asp Pro Val

325 330 335

Ala Val Ile Arg Thr His Ala Asn Arg Leu Gln Leu Ala Gly Val

340 345 350

<210> 6

<211> 364

<212> PRT

<213> Burkholderia foresta (Burkholderia arboris)

<400> 6

Met Ala Arg Ser Met Arg Ser Arg Val Met Ala Gly Ala Val Ala Cys

1 5 10 15

Ala Met Ser Val Ala Pro Phe Ala Gly Thr Thr Ala Leu Met Thr Leu

20 25 30

Ala Thr Thr His Ala Ala Met Ala Ala Thr Ala Pro Ala Asp Asn Tyr

35 40 45

Ala Ala Thr Arg Tyr Pro Ile Ile Leu Val His Gly Leu Thr Gly Thr

50 55 60

Asp Lys Tyr Ala Gly Val Leu Glu Tyr Trp Tyr Gly Ile Gln Glu Asp

65 70 75 80

Leu Gln Gln His Gly Ala Thr Val Tyr Val Ala Asn Leu Ser Gly Phe

85 90 95

Gln Ser Asp Asp Gly Pro Asn Gly Arg Gly Glu Gln Leu Leu Ala Tyr

100 105 110

Val Lys Thr Val Leu Ala Ala Thr Gly Ala Thr Lys Val Asn Leu Val

115 120 125

Gly His Ser Gln Gly Gly Leu Thr Ser Arg Tyr Val Ala Ala Val Ala

130 135 140

Pro Asp Leu Val Ala Ser Val Thr Thr Ile Gly Thr Pro His Arg Gly

145 150 155 160

Ser Glu Phe Ala Asp Phe Val Gln Ser Val Leu Ala Tyr Asp Pro Thr

165 170 175

Gly Leu Ser Ser Thr Val Ile Ala Ala Phe Val Asn Val Phe Gly Ile

180 185 190

Leu Thr Ser Ser Ser His Asn Thr Asn Gln Asp Ala Leu Ala Ser Leu

195 200 205

Lys Thr Leu Thr Thr Ala Gln Ala Ala Thr Tyr Asn Gln Asn Tyr Pro

210 215 220

Ser Ala Gly Leu Gly Ala Pro Gly Ser Cys Gln Thr Gly Ala Pro Thr

225 230 235 240

Glu Thr Val Gly Gly Asn Thr His Leu Leu Tyr Ser Trp Ala Gly Thr

245 250 255

Ala Ile Gln Pro Thr Leu Ser Leu Phe Gly Val Thr Gly Ala Lys Asp

260 265 270

Thr Ser Thr Ile Pro Leu Val Asp Pro Ala Asn Ala Leu Asp Pro Ser

275 280 285

Thr Leu Ala Leu Phe Gly Thr Gly Thr Val Met Ile Asn Arg Gly Ser

290 295 300

Gly Gln Asn Asp Gly Leu Val Ser Lys Cys Ser Ala Leu Tyr Gly Lys

305 310 315 320

Val Leu Ser Thr Ser Tyr Lys Trp Asn His Ile Asp Glu Ile Asn Gln

325 330 335

Leu Leu Gly Val Arg Gly Ala Tyr Ala Glu Asp Pro Val Ala Val Ile

340 345 350

Arg Thr His Ala Asn Arg Leu Gln Leu Ala Gly Val

355 360

<210> 7

<211> 1149

<212> DNA

<213> Burkholderia foresta (Burkholderia arboris)

<400> 7

aatgtcacct atcacgtcgc aggcatcccg accgccgtca ccgctcagca gttgctgtat 60

cgcaccaaca acgcgcagaa ccagcctgtc gtcaacgtga cgtcggtgat ccgcagccag 120

gtcagcaacg gccaggccat ttcgtaccag tcggcctacg attcgctgaa cccgtacgac 180

gagccgtcgc aggtgattgc cggcgaccgc gacgtgacca aggtcatcaa cgtcggcacg 240

ctgctctaca gtgcggagtc gatcccgctg tcgacgctgc tgctgctcgg ctacaacatc 300

atcgtgcccg atacggaagg ccagacggcg gacttcgcgg ccggccccga atacgggatg 360

acgacgctcg attcgatccg cgcggcgctt aatacgccgt cgacgggcct gaatccgtcg 420

agcaaggtcg cgatgatcgg ctattcgggc ggcgcaatcg cgacgaactg ggccgcgcag 480

ctcgcgccaa gttatgcgcc cgacatcaac aagcagctcg tcggcgcggc ggaaggcggc 540

gtgctggtcg atcccgcgca caacctgcgc tatgtcgacg gcagcatcgt gtggggcggc 600

gttgcggcgg ccgcgctggc cgggctgtcg cgcggctatg cgttcgacct gacgccgtat 660

ctcagcgata cgggcgtcgc cgtgttcaag gacatccaga accagtcgct tgcgtacatc 720

ctgccgaagt acacgggcct gcactggagc acgctgttca agccgcaata cgcgaacgac 780

atcaacagca tcccggcgta cgtgacgtat gcgaacaagg tgaacgcggg gctggccgca 840

tcgccgacga tcccgatgtt catcggccag ggcacggcag gcgcgctcga cggtaccttc 900

agcagccagg taggcgacgg cgtgatgctc gcgtacgacg tgcgcgccct ggcgcagaag 960

ttctgcgcca gcggcacgcc ggtcacgtac accgagtatc cgctggaaca tgcgggcgcg 1020

atcgtgccgt gggtggccgg gatgctgccc tggctctacg accgcttcaa cgggaaaacc 1080

gcgccgagca attgctggct gacgtcgctg ctgccgagca attcgctggc gcccgagacg 1140

ctgcactag 1149

<210> 8

<211> 1155

<212> DNA

<213> Burkholderia cerealis (Burkholderia andopogonis)

<400> 8

acgcgcaatg tcacctatca cgtcgcaggc atcccgaccg ccgtcaccgc tcagcagttg 60

ctgtatcgca ccaacaacgc gcagaaccag cctgtcgtca acgtgacgtc ggtgatccgc 120

agccaggtca gcaacggcca ggccatttcg taccagtcgg cctacgattc gctgaacccg 180

tacgacgagc cgtcgcaggt gattgccggc gaccgcgacg tgaccaaggt catcaacgtc 240

ggcacgctgc tctacagtgc ggagtcgatc ccgctgtcga cgctgctgct gctcggctac 300

aacatcatcg tgcccgatac ggaaggccag acggcggact tcgcggccgg ccccgaatac 360

gggatgacga cgctcgattc gatccgcgcg gcgcttaata cgccgtcgac gggcctgaat 420

ccgtcgagca aggtcgcgat gatcggctat tcgggcggcg caatcgcgac gaactgggcc 480

gcgcagctcg cgccaagtta tgcgcccgac atcaacaagc agctcgtcgg cgcggcggaa 540

ggcggcgtgc tggtcgatcc cgcgcacaac ctgcgctatg tcgacggcag catcgtgtgg 600

ggcggcgttg cggcggccgc gctggccggg ctgtcgcgcg gctatgcgtt cgacctgacg 660

ccgtatctca gcgatacggg cgtcgccgtg ttcaaggaca tccagaacca gtcgcttgcg 720

tacatcctgc cgaagtacac gggcctgcac tggagcacgc tgttcaagcc gcaatacgcg 780

aacgacatca acagcatccc ggcgtacgtg acgtatgcga acaaggtgaa cgcggggctg 840

gccgcatcgc cgacgatccc gatgttcatc ggccagggca cggcaggcgc gctcgacggt 900

accttcagca gccaggtagg cgacggcgtg atgctcgcgt acgacgtgcg cgccctggcg 960

cagaagttct gcgccagcgg cacgccggtc acgtacaccg agtatccgct ggaacatgcg 1020

ggcgcgatcg tgccgtgggt ggccgggatg ctgccctggc tctacgaccg cttcaacggg 1080

aaaaccgcgc cgagcaattg ctggctgacg tcgctgctgc cgagcaattc gctggcgccc 1140

gagacgctgc actag 1155

<210> 9

<211> 1239

<212> DNA

<213> Burkholderia foresta (Burkholderia arboris)

<400> 9

gccgcgccga ccgtgtccga tccgttctac acgtacaccg gcgccacgcc gctggcatcg 60

attccaccgg gcacggtgct gaagacgcgc aatgtcacct atcacgtcgc aggcatcccg 120

accgccgtca ccgctcagca gttgctgtat cgcaccaaca acgcgcagaa ccagcctgtc 180

gtcaacgtga cgtcggtgat ccgcagccag gtcagcaacg gccaggccat ttcgtaccag 240

tcggcctacg attcgctgaa cccgtacgac gagccgtcgc aggtgattgc cggcgaccgc 300

gacgtgacca aggtcatcaa cgtcggcacg ctgctctaca gtgcggagtc gatcccgctg 360

tcgacgctgc tgctgctcgg ctacaacatc atcgtgcccg atacggaagg ccagacggcg 420

gacttcgcgg ccggccccga atacgggatg acgacgctcg attcgatccg cgcggcgctt 480

aatacgccgt cgacgggcct gaatccgtcg agcaaggtcg cgatgatcgg ctattcgggc 540

ggcgcaatcg cgacgaactg ggccgcgcag ctcgcgccaa gttatgcgcc cgacatcaac 600

aagcagctcg tcggcgcggc ggaaggcggc gtgctggtcg atcccgcgca caacctgcgc 660

tatgtcgacg gcagcatcgt gtggggcggc gttgcggcgg ccgcgctggc cgggctgtcg 720

cgcggctatg cgttcgacct gacgccgtat ctcagcgata cgggcgtcgc cgtgttcaag 780

gacatccaga accagtcgct tgcgtacatc ctgccgaagt acacgggcct gcactggagc 840

acgctgttca agccgcaata cgcgaacgac atcaacagca tcccggcgta cgtgacgtat 900

gcgaacaagg tgaacgcggg gctggccgca tcgccgacga tcccgatgtt catcggccag 960

ggcacggcag gcgcgctcga cggtaccttc agcagccagg taggcgacgg cgtgatgctc 1020

gcgtacgacg tgcgcgccct ggcgcagaag ttctgcgcca gcggcacgcc ggtcacgtac 1080

accgagtatc cgctggaaca tgcgggcgcg atcgtgccgt gggtggccgg gatgctgccc 1140

tggctctacg accgcttcaa cgggaaaacc gcgccgagca attgctggct gacgtcgctg 1200

ctgccgagca attcgctggc gcccgagacg ctgcactag 1239

<210> 10

<211> 1299

<212> DNA

<213> Burkholderia foresta (Burkholderia arboris)

<400> 10

atgaccgtcg cggccgccgt gtgcgccgcg ctggccattg ccgcaccgtc ggccggcgcc 60

gccgcgccga ccgtgtccga tccgttctac acgtacaccg gcgccacgcc gctggcatcg 120

attccaccgg gcacggtgct gaagacgcgc aatgtcacct atcacgtcgc aggcatcccg 180

accgccgtca ccgctcagca gttgctgtat cgcaccaaca acgcgcagaa ccagcctgtc 240

gtcaacgtga cgtcggtgat ccgcagccag gtcagcaacg gccaggccat ttcgtaccag 300

tcggcctacg attcgctgaa cccgtacgac gagccgtcgc aggtgattgc cggcgaccgc 360

gacgtgacca aggtcatcaa cgtcggcacg ctgctctaca gtgcggagtc gatcccgctg 420

tcgacgctgc tgctgctcgg ctacaacatc atcgtgcccg atacggaagg ccagacggcg 480

gacttcgcgg ccggccccga atacgggatg acgacgctcg attcgatccg cgcggcgctt 540

aatacgccgt cgacgggcct gaatccgtcg agcaaggtcg cgatgatcgg ctattcgggc 600

ggcgcaatcg cgacgaactg ggccgcgcag ctcgcgccaa gttatgcgcc cgacatcaac 660

aagcagctcg tcggcgcggc ggaaggcggc gtgctggtcg atcccgcgca caacctgcgc 720

tatgtcgacg gcagcatcgt gtggggcggc gttgcggcgg ccgcgctggc cgggctgtcg 780

cgcggctatg cgttcgacct gacgccgtat ctcagcgata cgggcgtcgc cgtgttcaag 840

gacatccaga accagtcgct tgcgtacatc ctgccgaagt acacgggcct gcactggagc 900

acgctgttca agccgcaata cgcgaacgac atcaacagca tcccggcgta cgtgacgtat 960

gcgaacaagg tgaacgcggg gctggccgca tcgccgacga tcccgatgtt catcggccag 1020

ggcacggcag gcgcgctcga cggtaccttc agcagccagg taggcgacgg cgtgatgctc 1080

gcgtacgacg tgcgcgccct ggcgcagaag ttctgcgcca gcggcacgcc ggtcacgtac 1140

accgagtatc cgctggaaca tgcgggcgcg atcgtgccgt gggtggccgg gatgctgccc 1200

tggctctacg accgcttcaa cgggaaaacc gcgccgagca attgctggct gacgtcgctg 1260

ctgccgagca attcgctggc gcccgagacg ctgcactag 1299

<210> 11

<211> 382

<212> PRT

<213> Burkholderia foresta (Burkholderia arboris)

<400> 11

Asn Val Thr Tyr His Val Ala Gly Ile Pro Thr Ala Val Thr Ala Gln

1 5 10 15

Gln Leu Leu Tyr Arg Thr Asn Asn Ala Gln Asn Gln Pro Val Val Asn

20 25 30

Val Thr Ser Val Ile Arg Ser Gln Val Ser Asn Gly Gln Ala Ile Ser

35 40 45

Tyr Gln Ser Ala Tyr Asp Ser Leu Asn Pro Tyr Asp Glu Pro Ser Gln

50 55 60

Val Ile Ala Gly Asp Arg Asp Val Thr Lys Val Ile Asn Val Gly Thr

65 70 75 80

Leu Leu Tyr Ser Ala Glu Ser Ile Pro Leu Ser Thr Leu Leu Leu Leu

85 90 95

Gly Tyr Asn Ile Ile Val Pro Asp Thr Glu Gly Gln Thr Ala Asp Phe

100 105 110

Ala Ala Gly Pro Glu Tyr Gly Met Thr Thr Leu Asp Ser Ile Arg Ala

115 120 125

Ala Leu Asn Thr Pro Ser Thr Gly Leu Asn Pro Ser Ser Lys Val Ala

130 135 140

Met Ile Gly Tyr Ser Gly Gly Ala Ile Ala Thr Asn Trp Ala Ala Gln

145 150 155 160

Leu Ala Pro Ser Tyr Ala Pro Asp Ile Asn Lys Gln Leu Val Gly Ala

165 170 175

Ala Glu Gly Gly Val Leu Val Asp Pro Ala His Asn Leu Arg Tyr Val

180 185 190

Asp Gly Ser Ile Val Trp Gly Gly Val Ala Ala Ala Ala Leu Ala Gly

195 200 205

Leu Ser Arg Gly Tyr Ala Phe Asp Leu Thr Pro Tyr Leu Ser Asp Thr

210 215 220

Gly Val Ala Val Phe Lys Asp Ile Gln Asn Gln Ser Leu Ala Tyr Ile

225 230 235 240

Leu Pro Lys Tyr Thr Gly Leu His Trp Ser Thr Leu Phe Lys Pro Gln

245 250 255

Tyr Ala Asn Asp Ile Asn Ser Ile Pro Ala Tyr Val Thr Tyr Ala Asn

260 265 270

Lys Val Asn Ala Gly Leu Ala Ala Ser Pro Thr Ile Pro Met Phe Ile

275 280 285

Gly Gln Gly Thr Ala Gly Ala Leu Asp Gly Thr Phe Ser Ser Gln Val

290 295 300

Gly Asp Gly Val Met Leu Ala Tyr Asp Val Arg Ala Leu Ala Gln Lys

305 310 315 320

Phe Cys Ala Ser Gly Thr Pro Val Thr Tyr Thr Glu Tyr Pro Leu Glu

325 330 335

His Ala Gly Ala Ile Val Pro Trp Val Ala Gly Met Leu Pro Trp Leu

340 345 350

Tyr Asp Arg Phe Asn Gly Lys Thr Ala Pro Ser Asn Cys Trp Leu Thr

355 360 365

Ser Leu Leu Pro Ser Asn Ser Leu Ala Pro Glu Thr Leu His

370 375 380

<210> 12

<211> 384

<212> PRT

<213> Burkholderia foresta (Burkholderia arboris)

<400> 12

Thr Arg Asn Val Thr Tyr His Val Ala Gly Ile Pro Thr Ala Val Thr

1 5 10 15

Ala Gln Gln Leu Leu Tyr Arg Thr Asn Asn Ala Gln Asn Gln Pro Val

20 25 30

Val Asn Val Thr Ser Val Ile Arg Ser Gln Val Ser Asn Gly Gln Ala

35 40 45

Ile Ser Tyr Gln Ser Ala Tyr Asp Ser Leu Asn Pro Tyr Asp Glu Pro

50 55 60

Ser Gln Val Ile Ala Gly Asp Arg Asp Val Thr Lys Val Ile Asn Val

65 70 75 80

Gly Thr Leu Leu Tyr Ser Ala Glu Ser Ile Pro Leu Ser Thr Leu Leu

85 90 95

Leu Leu Gly Tyr Asn Ile Ile Val Pro Asp Thr Glu Gly Gln Thr Ala

100 105 110

Asp Phe Ala Ala Gly Pro Glu Tyr Gly Met Thr Thr Leu Asp Ser Ile

115 120 125

Arg Ala Ala Leu Asn Thr Pro Ser Thr Gly Leu Asn Pro Ser Ser Lys

130 135 140

Val Ala Met Ile Gly Tyr Ser Gly Gly Ala Ile Ala Thr Asn Trp Ala

145 150 155 160

Ala Gln Leu Ala Pro Ser Tyr Ala Pro Asp Ile Asn Lys Gln Leu Val

165 170 175

Gly Ala Ala Glu Gly Gly Val Leu Val Asp Pro Ala His Asn Leu Arg

180 185 190

Tyr Val Asp Gly Ser Ile Val Trp Gly Gly Val Ala Ala Ala Ala Leu

195 200 205

Ala Gly Leu Ser Arg Gly Tyr Ala Phe Asp Leu Thr Pro Tyr Leu Ser

210 215 220

Asp Thr Gly Val Ala Val Phe Lys Asp Ile Gln Asn Gln Ser Leu Ala

225 230 235 240

Tyr Ile Leu Pro Lys Tyr Thr Gly Leu His Trp Ser Thr Leu Phe Lys

245 250 255

Pro Gln Tyr Ala Asn Asp Ile Asn Ser Ile Pro Ala Tyr Val Thr Tyr

260 265 270

Ala Asn Lys Val Asn Ala Gly Leu Ala Ala Ser Pro Thr Ile Pro Met

275 280 285

Phe Ile Gly Gln Gly Thr Ala Gly Ala Leu Asp Gly Thr Phe Ser Ser

290 295 300

Gln Val Gly Asp Gly Val Met Leu Ala Tyr Asp Val Arg Ala Leu Ala

305 310 315 320

Gln Lys Phe Cys Ala Ser Gly Thr Pro Val Thr Tyr Thr Glu Tyr Pro

325 330 335

Leu Glu His Ala Gly Ala Ile Val Pro Trp Val Ala Gly Met Leu Pro

340 345 350

Trp Leu Tyr Asp Arg Phe Asn Gly Lys Thr Ala Pro Ser Asn Cys Trp

355 360 365

Leu Thr Ser Leu Leu Pro Ser Asn Ser Leu Ala Pro Glu Thr Leu His

370 375 380

<210> 13

<211> 412

<212> PRT

<213> Burkholderia foresta (Burkholderia arboris)

<400> 13

Ala Ala Pro Thr Val Ser Asp Pro Phe Tyr Thr Tyr Thr Gly Ala Thr

1 5 10 15

Pro Leu Ala Ser Ile Pro Pro Gly Thr Val Leu Lys Thr Arg Asn Val

20 25 30

Thr Tyr His Val Ala Gly Ile Pro Thr Ala Val Thr Ala Gln Gln Leu

35 40 45

Leu Tyr Arg Thr Asn Asn Ala Gln Asn Gln Pro Val Val Asn Val Thr

50 55 60

Ser Val Ile Arg Ser Gln Val Ser Asn Gly Gln Ala Ile Ser Tyr Gln

65 70 75 80

Ser Ala Tyr Asp Ser Leu Asn Pro Tyr Asp Glu Pro Ser Gln Val Ile

85 90 95

Ala Gly Asp Arg Asp Val Thr Lys Val Ile Asn Val Gly Thr Leu Leu

100 105 110

Tyr Ser Ala Glu Ser Ile Pro Leu Ser Thr Leu Leu Leu Leu Gly Tyr

115 120 125

Asn Ile Ile Val Pro Asp Thr Glu Gly Gln Thr Ala Asp Phe Ala Ala

130 135 140

Gly Pro Glu Tyr Gly Met Thr Thr Leu Asp Ser Ile Arg Ala Ala Leu

145 150 155 160

Asn Thr Pro Ser Thr Gly Leu Asn Pro Ser Ser Lys Val Ala Met Ile

165 170 175

Gly Tyr Ser Gly Gly Ala Ile Ala Thr Asn Trp Ala Ala Gln Leu Ala

180 185 190

Pro Ser Tyr Ala Pro Asp Ile Asn Lys Gln Leu Val Gly Ala Ala Glu

195 200 205

Gly Gly Val Leu Val Asp Pro Ala His Asn Leu Arg Tyr Val Asp Gly

210 215 220

Ser Ile Val Trp Gly Gly Val Ala Ala Ala Ala Leu Ala Gly Leu Ser

225 230 235 240

Arg Gly Tyr Ala Phe Asp Leu Thr Pro Tyr Leu Ser Asp Thr Gly Val

245 250 255

Ala Val Phe Lys Asp Ile Gln Asn Gln Ser Leu Ala Tyr Ile Leu Pro

260 265 270

Lys Tyr Thr Gly Leu His Trp Ser Thr Leu Phe Lys Pro Gln Tyr Ala

275 280 285

Asn Asp Ile Asn Ser Ile Pro Ala Tyr Val Thr Tyr Ala Asn Lys Val

290 295 300

Asn Ala Gly Leu Ala Ala Ser Pro Thr Ile Pro Met Phe Ile Gly Gln

305 310 315 320

Gly Thr Ala Gly Ala Leu Asp Gly Thr Phe Ser Ser Gln Val Gly Asp

325 330 335

Gly Val Met Leu Ala Tyr Asp Val Arg Ala Leu Ala Gln Lys Phe Cys

340 345 350

Ala Ser Gly Thr Pro Val Thr Tyr Thr Glu Tyr Pro Leu Glu His Ala

355 360 365

Gly Ala Ile Val Pro Trp Val Ala Gly Met Leu Pro Trp Leu Tyr Asp

370 375 380

Arg Phe Asn Gly Lys Thr Ala Pro Ser Asn Cys Trp Leu Thr Ser Leu

385 390 395 400

Leu Pro Ser Asn Ser Leu Ala Pro Glu Thr Leu His

405 410

<210> 14

<211> 432

<212> PRT

<213> Burkholderia foresta (Burkholderia arboris)

<400> 14

Met Thr Val Ala Ala Ala Val Cys Ala Ala Leu Ala Ile Ala Ala Pro

1 5 10 15

Ser Ala Gly Ala Ala Ala Pro Thr Val Ser Asp Pro Phe Tyr Thr Tyr

20 25 30

Thr Gly Ala Thr Pro Leu Ala Ser Ile Pro Pro Gly Thr Val Leu Lys

35 40 45

Thr Arg Asn Val Thr Tyr His Val Ala Gly Ile Pro Thr Ala Val Thr

50 55 60

Ala Gln Gln Leu Leu Tyr Arg Thr Asn Asn Ala Gln Asn Gln Pro Val

65 70 75 80

Val Asn Val Thr Ser Val Ile Arg Ser Gln Val Ser Asn Gly Gln Ala

85 90 95

Ile Ser Tyr Gln Ser Ala Tyr Asp Ser Leu Asn Pro Tyr Asp Glu Pro

100 105 110

Ser Gln Val Ile Ala Gly Asp Arg Asp Val Thr Lys Val Ile Asn Val

115 120 125

Gly Thr Leu Leu Tyr Ser Ala Glu Ser Ile Pro Leu Ser Thr Leu Leu

130 135 140

Leu Leu Gly Tyr Asn Ile Ile Val Pro Asp Thr Glu Gly Gln Thr Ala

145 150 155 160

Asp Phe Ala Ala Gly Pro Glu Tyr Gly Met Thr Thr Leu Asp Ser Ile

165 170 175

Arg Ala Ala Leu Asn Thr Pro Ser Thr Gly Leu Asn Pro Ser Ser Lys

180 185 190

Val Ala Met Ile Gly Tyr Ser Gly Gly Ala Ile Ala Thr Asn Trp Ala

195 200 205

Ala Gln Leu Ala Pro Ser Tyr Ala Pro Asp Ile Asn Lys Gln Leu Val

210 215 220

Gly Ala Ala Glu Gly Gly Val Leu Val Asp Pro Ala His Asn Leu Arg

225 230 235 240

Tyr Val Asp Gly Ser Ile Val Trp Gly Gly Val Ala Ala Ala Ala Leu

245 250 255

Ala Gly Leu Ser Arg Gly Tyr Ala Phe Asp Leu Thr Pro Tyr Leu Ser

260 265 270

Asp Thr Gly Val Ala Val Phe Lys Asp Ile Gln Asn Gln Ser Leu Ala

275 280 285

Tyr Ile Leu Pro Lys Tyr Thr Gly Leu His Trp Ser Thr Leu Phe Lys

290 295 300

Pro Gln Tyr Ala Asn Asp Ile Asn Ser Ile Pro Ala Tyr Val Thr Tyr

305 310 315 320

Ala Asn Lys Val Asn Ala Gly Leu Ala Ala Ser Pro Thr Ile Pro Met

325 330 335

Phe Ile Gly Gln Gly Thr Ala Gly Ala Leu Asp Gly Thr Phe Ser Ser

340 345 350

Gln Val Gly Asp Gly Val Met Leu Ala Tyr Asp Val Arg Ala Leu Ala

355 360 365

Gln Lys Phe Cys Ala Ser Gly Thr Pro Val Thr Tyr Thr Glu Tyr Pro

370 375 380

Leu Glu His Ala Gly Ala Ile Val Pro Trp Val Ala Gly Met Leu Pro

385 390 395 400

Trp Leu Tyr Asp Arg Phe Asn Gly Lys Thr Ala Pro Ser Asn Cys Trp

405 410 415

Leu Thr Ser Leu Leu Pro Ser Asn Ser Leu Ala Pro Glu Thr Leu His

420 425 430

<210> 15

<211> 963

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> modified sequence of the 1 st lipase

<400> 15

tcggacgact acgcgacgac gcgttatccg atcatcctcg tgcacgggct cacgggcacc 60

gacaagtacg cgggcgtgct cgagtactgg tacggcatcc aggaagacct gcagcagcat 120

ggcgcgaccg tctacgtcgc gaacctgtcg ggcttccaga gcgatgacgg cccgaacggg 180

cgcggcgaac agctgctcgc ttacgtgaag acggtgctcg ccgcgacggg cgcgaccaag 240

gtcaatctcg tcggtcacag ccagggcggg ctcacgtcgc gttatgtcgc ggccgtcgcg 300

cccgatctcg tcgcgtcggt gacgacgatc ggcacgccgc atcgcggctc cgagttcgcc 360

gacttcgtgc agagcgtgct cgcatacgat ccgaccgggc tttcgtcgtc ggtgatcgcc 420

gcgttcgtca atgtgttcgg aatcctgacg agcagcagtc acaacacgaa ccaggacgcg 480

ctcgcgtcgc tgaagacgct gacgaccgca caggccgcca cgtacaacca gaactatccg 540

agcgcgggcc ttggcgcgcc gggcagttgc cagaccggcg caccgacgga aaccgtcggc 600

ggcaacacgc atctgctgta ttcgtgggcc ggcacggcga tccagccgac gctctccgtg 660

ttcggtgtca cgggtgcgac ggacacgagc accattccgc tcgtcgaccc ggcgaacgcg 720

ctcgatctgt cgacgctcgc gctgttcggc acgggcacgg tgatgatcaa ccgcggttcg 780

ggccagaacg acgggctcgt gtcgaagtgc agcgcgctgt acggccaggt gctgagcacg 840

agctacaagt ggaaccatat cgacgagatc aaccagttgc ttggcgtgcg cggcgcgtat 900

gcggaagatc cggtcgcggt gatccgcacg catgcgaacc ggctgaagct cgcgggcgtg 960

taa 963

<210> 16

<211> 320

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> modified sequence of the 1 st lipase

<400> 16

Ser Asp Asp Tyr Ala Thr Thr Arg Tyr Pro Ile Ile Leu Val His Gly

1 5 10 15

Leu Thr Gly Thr Asp Lys Tyr Ala Gly Val Leu Glu Tyr Trp Tyr Gly

20 25 30

Ile Gln Glu Asp Leu Gln Gln His Gly Ala Thr Val Tyr Val Ala Asn

35 40 45

Leu Ser Gly Phe Gln Ser Asp Asp Gly Pro Asn Gly Arg Gly Glu Gln

50 55 60

Leu Leu Ala Tyr Val Lys Thr Val Leu Ala Ala Thr Gly Ala Thr Lys

65 70 75 80

Val Asn Leu Val Gly His Ser Gln Gly Gly Leu Thr Ser Arg Tyr Val

85 90 95

Ala Ala Val Ala Pro Asp Leu Val Ala Ser Val Thr Thr Ile Gly Thr

100 105 110

Pro His Arg Gly Ser Glu Phe Ala Asp Phe Val Gln Ser Val Leu Ala

115 120 125

Tyr Asp Pro Thr Gly Leu Ser Ser Ser Val Ile Ala Ala Phe Val Asn

130 135 140

Val Phe Gly Ile Leu Thr Ser Ser Ser His Asn Thr Asn Gln Asp Ala

145 150 155 160

Leu Ala Ser Leu Lys Thr Leu Thr Thr Ala Gln Ala Ala Thr Tyr Asn

165 170 175

Gln Asn Tyr Pro Ser Ala Gly Leu Gly Ala Pro Gly Ser Cys Gln Thr

180 185 190

Gly Ala Pro Thr Glu Thr Val Gly Gly Asn Thr His Leu Leu Tyr Ser

195 200 205

Trp Ala Gly Thr Ala Ile Gln Pro Thr Leu Ser Val Phe Gly Val Thr

210 215 220

Gly Ala Thr Asp Thr Ser Thr Ile Pro Leu Val Asp Pro Ala Asn Ala

225 230 235 240

Leu Asp Leu Ser Thr Leu Ala Leu Phe Gly Thr Gly Thr Val Met Ile

245 250 255

Asn Arg Gly Ser Gly Gln Asn Asp Gly Leu Val Ser Lys Cys Ser Ala

260 265 270

Leu Tyr Gly Gln Val Leu Ser Thr Ser Tyr Lys Trp Asn His Ile Asp

275 280 285

Glu Ile Asn Gln Leu Leu Gly Val Arg Gly Ala Tyr Ala Glu Asp Pro

290 295 300

Val Ala Val Ile Arg Thr His Ala Asn Arg Leu Lys Leu Ala Gly Val

305 310 315 320

<210> 17

<211> 1149

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> modified sequence of 2nd lipase

<400> 17

aacgtcacct atcacgtggc cggcattccg accgcgctga ccgcgcagca gttgctgtat 60

cgcaccaata acgcgctgaa ccagccggtc gtgaacgtga cgtcggtgat ccggagccag 120

gtcagcaacg gccgggccat ctcgtaccag tcggcttacg attcgctgaa cccgtacgac 180

gagccgtcgc aggtgatcgc cggcgatcgc gacgtcacga agatcatcaa cgtcggcacg 240

ttgctctaca gcgcggaatc gattccgctg tcgacgctgc tgctgctcgg ctacaacgtc 300

atcgtgcccg atacggaagg ccagacggcc gatttcgccg ccggccccga atacgggatg 360

acgacgctcg attcgatccg cgcggcgctc aatacgccgt cgaccggcct gagtccgtcg 420

agcaaggtcg cgatgatcgg ctattccggc ggcgcgatcg cgacgaactg ggccgcgcag 480

ctcgcgccga gctatgcacc cgagatcaac aggcagctcg tcggcgcggc ggagggcggc 540

gtgctggtcg accccgcgca caacctgcgc tatgtcgacg gcagcatcgt gtggggcggc 600

gtggccgcgg ccgcgctggc cgggctgtcg cgcggctacg gcttcgacct gacgccctat 660

ctcagcgata ccggcgtcgc cgtgttcaac gacatccaga gccagtcgct cgcgtacatc 720

ctgccgaaat acacggggct gcgctggggc acgctgttca agccgcaata cgcgaacgac 780

atcaacagca ttcccgcgta cgtgacgtat gccaacaagg tgaatgccgg gctggccgca 840

tcgccgacga tcccgatgtt catcggccag ggcacggcgg gcgcgctgga cggcaccttc 900

agcagccagg tgggtgacgg cgtgatgctc gcgtatgacg tgcgcgccct tgcgcagaag 960

ttctgcgcca gcggcacgcc ggtcacgtac aacgagtatc cgctcgagca tgcgggggcg 1020

atcgtgccgt gggtggccgg catgctgccg tggctctacg accgtttcaa cgggaaagcc 1080

gcgccgagca attgctggct gacgtcgctg ctgccgagca attcgctggc gcccgaaacg 1140

ctgcattag 1149

<210> 18

<211> 382

<212> PRT

<213> Artificial sequence (artificial sequence)

<220>

<223> modified sequence of 2nd lipase

<400> 18

Asn Val Thr Tyr His Val Ala Gly Ile Pro Thr Ala Leu Thr Ala Gln

1 5 10 15

Gln Leu Leu Tyr Arg Thr Asn Asn Ala Leu Asn Gln Pro Val Val Asn

20 25 30

Val Thr Ser Val Ile Arg Ser Gln Val Ser Asn Gly Arg Ala Ile Ser

35 40 45

Tyr Gln Ser Ala Tyr Asp Ser Leu Asn Pro Tyr Asp Glu Pro Ser Gln

50 55 60

Val Ile Ala Gly Asp Arg Asp Val Thr Lys Ile Ile Asn Val Gly Thr

65 70 75 80

Leu Leu Tyr Ser Ala Glu Ser Ile Pro Leu Ser Thr Leu Leu Leu Leu

85 90 95

Gly Tyr Asn Val Ile Val Pro Asp Thr Glu Gly Gln Thr Ala Asp Phe

100 105 110

Ala Ala Gly Pro Glu Tyr Gly Met Thr Thr Leu Asp Ser Ile Arg Ala

115 120 125

Ala Leu Asn Thr Pro Ser Thr Gly Leu Ser Pro Ser Ser Lys Val Ala

130 135 140

Met Ile Gly Tyr Ser Gly Gly Ala Ile Ala Thr Asn Trp Ala Ala Gln

145 150 155 160

Leu Ala Pro Ser Tyr Ala Pro Glu Ile Asn Arg Gln Leu Val Gly Ala

165 170 175

Ala Glu Gly Gly Val Leu Val Asp Pro Ala His Asn Leu Arg Tyr Val

180 185 190

Asp Gly Ser Ile Val Trp Gly Gly Val Ala Ala Ala Ala Leu Ala Gly

195 200 205

Leu Ser Arg Gly Tyr Gly Phe Asp Leu Thr Pro Tyr Leu Ser Asp Thr

210 215 220

Gly Val Ala Val Phe Asn Asp Ile Gln Ser Gln Ser Leu Ala Tyr Ile

225 230 235 240

Leu Pro Lys Tyr Thr Gly Leu Arg Trp Gly Thr Leu Phe Lys Pro Gln

245 250 255

Tyr Ala Asn Asp Ile Asn Ser Ile Pro Ala Tyr Val Thr Tyr Ala Asn

260 265 270

Lys Val Asn Ala Gly Leu Ala Ala Ser Pro Thr Ile Pro Met Phe Ile

275 280 285

Gly Gln Gly Thr Ala Gly Ala Leu Asp Gly Thr Phe Ser Ser Gln Val

290 295 300

Gly Asp Gly Val Met Leu Ala Tyr Asp Val Arg Ala Leu Ala Gln Lys

305 310 315 320

Phe Cys Ala Ser Gly Thr Pro Val Thr Tyr Asn Glu Tyr Pro Leu Glu

325 330 335

His Ala Gly Ala Ile Val Pro Trp Val Ala Gly Met Leu Pro Trp Leu

340 345 350

Tyr Asp Arg Phe Asn Gly Lys Ala Ala Pro Ser Asn Cys Trp Leu Thr

355 360 365

Ser Leu Leu Pro Ser Asn Ser Leu Ala Pro Glu Thr Leu His

370 375 380