Means and method for cracking zearalenone
阅读说明：本技术 用于裂解玉米赤霉烯酮的手段和方法 (Means and method for cracking zearalenone ) 是由 M·萨姆海塞尔 S·弗鲁豪夫 W·D·摩尔 A·霍巴特纳 G·沙特兹迈尔 E·M·宾德 于 2019-07-30 设计创作，主要内容包括：本发明涉及一种用于增加α/β-水解酶的稳定性的方法。另外,本发明还涉及一种通过本发明的方法能够获得的α/β-水解酶。本发明还提供具有降低的总平均亲水性(GRAVY)值和/或包含特定突变的α/β-水解酶。此外,本发明涉及本发明的α/β-水解酶用于降解玉米赤霉烯酮(ZEN)的用途。(The present invention relates to a method for increasing the stability of α/β -hydrolase, additionally, the present invention also relates to a α/β -hydrolase obtainable by the method of the present invention also provides α/β -hydrolase having a reduced overall average hydrophilicity (GRAVY) value and/or comprising specific mutations furthermore, the present invention relates to the use of the α/β -hydrolase of the present invention for degrading Zearalenone (ZEN).)
1. A method for increasing the stability of α/β -hydrolase, the α/β -hydrolase comprising a sequence corresponding to positions 145 through 218 of SEQ ID NO:1, or a sequence having 58% or greater sequence identity to a sequence corresponding to positions 145 through 218 of SEQ ID NO:1, the method comprising:
replacing at least one amino acid at the following positions:
-positions corresponding to positions 160 to 205 of SEQ ID NO:1, or
Positions corresponding to positions 159 to 204 of SEQ ID NO 2, or
-positions corresponding to positions 160 to 205 of SEQ ID NO 3,4 or 5, or
Positions corresponding to positions 176 to 222 of SEQ ID NO 6,
wherein the amino acid is replaced with an amino acid that: the amino acids used have a more negative hydrophilicity index than the amino acids being replaced,
wherein the hydropathic index is determined by Kyte and Doolittle hydropathic index,
α/β -hydrolase having increased stability is thus obtained.
2. The method of claim 1, wherein the method comprises substituting at least one amino acid at the following positions:
positions corresponding to positions 185 to 191 of SEQ ID NO.1, and/or
Positions corresponding to positions 184 to 190 of SEQ ID NO 2, and/or
Positions corresponding to positions 185 to 191 of SEQ ID NO 3,4 or 5, and/or
Positions corresponding to positions 201 to 208 of SEQ ID NO 6.
3. The method according to claim 1 or 2, wherein the amino acid is substituted with an amino acid selected from R, K, N, Q, D, E, H, P, Y, W, S, T, G, A, M, C, F, L or V, preferably the amino acid used is selected from R, K, N, Q, D, E, H, P, Y, W, S, T or G, more preferably from R, K, N, Q, D, E, H or P.
4. The method according to any one of the preceding claims, wherein the amino acid is substituted with an amino acid selected from R, D, H, G, N or P, preferably R, D, H, G or N, more preferably the amino acid used is selected from R or N.
5. The method of any one of the preceding claims, wherein the amino acid substitution is selected from one or more of: v → A, G → R, G → S, A → P, A → R, A → D, A → H, A → N, A → G, S → D, P → H, M → D, G → E, I → A, I → V, H → N, Q → K, F → Y and/or V → C, preferably, the amino acid substitution is selected from one or more of: V160A, G185R, G185S, a186P, a186R, a188D, a188H, a188N, a188G, a188R, S189D, P190H, M191D, G199E, I200A, I200V, H203N, Q205K, F183Y and/or V197C, more preferably the amino acid substitution is selected from G185R, a186R, a188R, a188D, a188H, a188N and/or M191D.
6. The method of any one of the preceding claims, wherein the increased stability is a decrease in GRAVY value, an increase in pH stability and/or an increase in temperature stability.
7. An α/β -hydrolase obtainable by the method of any one of claims 4,5 or 6.
8. An α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO:1 or a sequence having greater than 58% sequence identity to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1,
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to positions 160 to 205 of SEQ ID NO:1, or
Positions corresponding to positions 159 to 204 of SEQ ID NO 2, or
-positions corresponding to positions 160 to 205 of SEQ ID NO 3,4 or 5, or
Positions corresponding to positions 176 to 222 of SEQ ID NO 6,
wherein said α/β -hydrolase has a GRAVY value that is at least 0.6% negative compared to the GRAVY value of a α/β -hydrolase having the polypeptide sequence of SEQ ID NO: 1.
9. An α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO:1 or a sequence having greater than 58% sequence identity to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1,
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to positions 185 to 191 of SEQ ID NO 1, or
Positions corresponding to positions 184 to 190 of SEQ ID NO 2, or
-positions corresponding to positions 185 to 191 of SEQ ID NO 3,4 or 5, or
Positions corresponding to positions 201 to 208 of SEQ ID NO 6,
wherein the amino acid substitution is selected from V → A, G → R, G → S, A → P, A → R, A → D, A → H, A → N, A → G, S → D, P → H, M → D, G → E, I → A, I → V, H → N and Q → K, and/or
Wherein the amino acid is replaced with an amino acid selected from P, R, D, H, G or N, preferably the amino acid used is selected from R, D, H, G or N, more preferably the amino acid used is selected from R or N.
10. An α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO 6 or a sequence having greater than 58% sequence identity to a sequence corresponding to positions 161 to 235 of SEQ ID NO 6,
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to positions 160 to 205 of SEQ ID NO:1, or
Positions corresponding to positions 159 to 204 of SEQ ID NO 2, or
-positions corresponding to positions 160 to 205 of SEQ ID NO 3,4 or 5, or
Positions corresponding to positions 176 to 222 of SEQ ID NO 6,
wherein said α/β -hydrolase has a GRAVY value that is at least 0.6% negative compared to the GRAVY value of a α/β -hydrolase having the polypeptide sequence of SEQ ID NO 6.
11. The α/β -hydrolase of any one of the preceding claims, wherein the α/β -hydrolase comprises the following amino acid substitutions:
-G → R and a → N, preferably G185R and a 188N;
-G → S and a → R, preferably G185S and a 188R;
-G → R, A → R, A → H, S → D, P → H and M → D, preferably G185R, a186R, a188H, S189D, P190H and M191D;
-V → A, G → R, A → N, G → E, I → A, H → N and Q → K, preferably V160A, G185R, a188N, G199E, I200A, H203N and Q205K;
-V → A, G → S, A → R, G → E, I → A, H → N and Q → K, preferably V160A, G185S, a188R, G199E, I200A, H203N and Q205K;
-V → A, G → E, I → A, H → N and Q → K, preferably V160A, G199E, I200A, H203N and Q205K;
-V → A, G → R, A → R, A → H, G → E, I → V, H → N and Q → K, preferably V160A, G185R, a186R, a188H, G199E, I200V, H203N and Q205K;
-V → A, G → R, A → R, A → H, S → D, P → H, M → D, G → E, I → V, H → N and Q → K, preferably V160A, G185R, a186R, a188H, S189D, P190H, M191D, G199E, I200V, H203N and Q205K; and/or
F → Y and V → C, preferably F183Y and V197C.
12. Use of the α/β -hydrolase of any of the preceding claims for degrading Zearalenone (ZEN).
13. A composition comprising the α/β -hydrolase of any one of the preceding claims, preferably the composition is a food or feed additive or a food or feed product.
14. The α/β -hydrolase or the composition of any one of the preceding claims, for use in treating or preventing a disease.
15. A kit comprising the α/β -hydrolase or the composition of any one of the preceding claims.
Technical Field
The present invention relates to a method for increasing the stability of α/β -hydrolase additionally, the present invention relates to a α/β -hydrolase obtainable by the method of the present invention also provides α/β -hydrolase having a reduced overall average hydrophilicity (GRAVY) value and/or comprising specific mutations furthermore the present invention relates to the use of the α/β -hydrolase of the present invention for degrading Zearalenone (ZEN).
Background
Mycotoxins are secondary metabolites produced by filamentous fungi. An important representative of mycotoxins is Zearalenone (ZEN), previously known as F-2 toxin, produced by many fusarium fungi and found throughout the world. These fungi in particular infect cultivated plants, such as various cereals, wherein fungal infection usually occurs before harvest, when fungal growth and/or mycotoxin production can occur before storage or even after harvest, i.e. before storage or under inappropriate storage conditions. The united nations Food and Agriculture Organization (FAO) estimates that 25% of agricultural products are contaminated with mycotoxins worldwide, which results in significant economic losses. In an international study spanning 8 years, a total of 19,757 samples were analyzed from
ZEN binds to estrogen receptors and can cause hormonal disturbances, is immediately absorbed after oral ingestion, and is converted by mammals into two stereoisomeric metabolites, α -zearalenol (α -ZEL) and/or β -zearalenol (β -ZEL), respectively, for example, α -ZEL and α -zearalanol (α -ZAL) and/or Zearalenone (ZAN) have a stronger estrogenic effect than ZEN although conjugated ZEN derivatives have lower estrogenic activity than ZEN itself, ZEN can be released again from these conjugated ZEN derivatives in the digestive tract, thereby restoring its full estrogenic activity.
ZEN has oral LD50 of up to 20,000mg/kg body weight and can exhibit sub-acute and/or sub-chronic toxic effects such as teratogenic, carcinogenic, estrogenic and immunosuppressive effects in chronically exposed animals or humans. The ZEN contaminated feed resulted in developmental disorders in mammals. Wherein pigs, especially piglets, are extremely sensitive to ZEN. Concentrations of ZEN above 0.5ppm in the feed lead to developmental disorders, concentrations above 1.5ppm can lead to hyperaestrogenicity in pigs. In cattle, a concentration of 12ppmZEN can cause spontaneous abortion.
Immediate and quantitative inactivation is necessary because of the rapid absorption through the mucosa, in particular through the gastric mucosa as well as the oral mucosa ZEN. Can be detected in the blood already 30 minutes after oral administration of ZEN. Due to the detrimental effects of ZEN, the european union has a legal upper limit on ZEN in food products and recommendations for an upper limit on ZEN in feed (EC No. 1881/2006).
The initial strategy for reducing ZEN contamination of food and feed products was to limit fungal growth, for example by following "good agricultural norms". This includes, inter alia, ensuring that the seeds are not infested with pests and fungi, or ensuring that agricultural waste is removed from the field in a timely manner. In addition, fungal growth in the field can be reduced by the use of fungicides. After harvesting, the harvested material should be stored at a residual moisture of less than 15% and at low temperature to prevent fungal growth. Likewise, material contaminated by fungal infestation should be removed prior to further processing. Despite this series of preventive measures, even in regions with the highest agricultural standards (such as north america and central europe), up to 37% of the test corn samples were found to be contaminated with ZEN in 2004 to 2011 (Schatzmayr and Streit (2013)).
Disclosure of Invention
In order to overcome the above problems and disadvantages, there is a need to develop another α/β -hydrolase, which α/β -hydrolase is capable of detoxifying ZEN and is suitable for use as a food or feed additive or food or feed product.
Aspects of the invention are described below, illustrated in embodiments, shown in the drawings, and reflected in the claims.
The present invention relates to a method for increasing the stability of α/β -hydrolase, said α/β -hydrolase comprising a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, or a sequence having 58% or more sequence identity to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1 (CAP-domain; 58% identity to CAP-domain of SEQ ID NO: 1), said method comprising:
replacing at least one amino acid at the following positions:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein the amino acid is substituted with an amino acid having a more negative hydropathic index than the substituted amino acid, wherein the hydropathic index is determined by Kyte and Doolittle hydropathic indices, thereby obtaining α/β -hydrolase having increased stability.
Furthermore, the present invention relates to an α/β -hydrolase obtainable by the process of the invention.
The invention also provides an α/β -hydrolase having a polypeptide sequence comprising a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, or a sequence having greater than 58% sequence identity to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1,
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein said α/β -hydrolase has a total average hydrophilicity (GRAVY) value that is at least 0.6% negative compared to the GRAVY value of a α/β -hydrolase having a polypeptide sequence of SEQ ID NO: 1.
The invention also relates to an α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO:1 or a sequence having more than 58% sequence identity to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1,
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 184 to 190 of
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 201 to 208 of
wherein the amino acid substitution is selected from V → A, G → R, G → S, A → P, A → R, A → D, A → H, A → N, A → G, S → D, P → H, M → D, G → E, I → A, I → V, H → N and Q → K, and/or
Wherein the amino acid is replaced with an amino acid selected from P, R, D, H, G or N, preferably the amino acid used is selected from R, D, H, G or N, more preferably the amino acid used is selected from R or N.
The invention also relates to an α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO. 6 or a sequence having more than 58% sequence identity to a sequence corresponding to positions 161 to 235 of SEQ ID NO. 6,
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein said α/β -hydrolase has a GRAVY value that is at least 0.6% negative compared to the GRAVY value of a α/β -hydrolase having the polypeptide sequence of
Furthermore, the present invention relates to the use of the α/β -hydrolase of the invention for degrading ZEN.
Further, the present invention relates to a composition comprising the α/β -hydrolase of the invention, preferably the composition is a food or feed additive or a food or feed product.
In addition, the present invention relates to the α/β -hydrolase or the composition of the invention for use in the treatment or prevention of a disease.
Further, the present invention relates to a kit comprising the α/β -hydrolase or the composition of the present invention.
Drawings
FIG. 1: the position of the CAP-domain, VI-domain and CAP-loop. 1-6, CAP-domain, VI-domain and CAP-loop amino acid positions.
FIGS. 2A-2G: different mutations in the VI-domain and/or the CAP-loop and their effect on the GRAVY value. 2A: the effect of modifications in the VI-domain of SEQ ID NO.1 on the GRAVY value of the variant of SEQ ID NO. 1. 2B: the effect of modifications in the VI-domain of SEQ ID NO:1 on the GRAVY value of the CAP-domain of the SEQ ID NO:1 variant. 2C: the effect of modifications in the VI-domain of SEQ ID NO.1 on the GRAVY value of the VI-domain of the variant of SEQ ID NO. 1. 2D: the effect of modifications in the CAP-loop of SEQ ID NO:1 on the GRAVY value of the CAP-loop of the SEQ ID NO:1 variant. 2E: the effect of modifications in the VI-domain of SEQ ID NO. 6 on the GRAVY values of the variants of SEQ ID NO. 6. 2F: the effect of modifications in the VI-domain of SEQ ID NO. 6 on the GRAVY value of the VI-domain of the variant SEQ ID NO. 6. 2G: the effect of modifications in the VI-domain of SEQ ID NO. 6 on the GRAVY value of the VI-domain of the variant SEQ ID NO. 6.
FIGS. 3A-3B: the percentage increase in temperature stability of ZEN degrading polypeptides relative to the polypeptides
FIG. 4: activity of polypeptide variants that degrade ZEN after incubation at pH4.0 compared to activity after incubation at pH7.5 (═ pH stability). Residual percent activity of ZEN-degrading polypeptide variants after incubation at pH4.0 compared to the same polypeptide variants after incubation at pH 7.5. The residual activity (pH stability) of the parent polypeptide SEQ ID NO.1 was 2.5%.
FIG. 5: 6500 selected response monitoring parameters on QTrap for analysis of samples from pig feeding trials. Analysis of samples from the pig feeding trial was performed on an Agilent 1290 series UHPLC system connected to a 6500QTrap mass spectrometer. Selected reaction monitoring parameters are shown. The product ions are given in quantitative (quatifier)/qualitative (qualifier).
Figure 6 analysis results of urine samples from pig feeding trials compared to SEQ ID NO:1 total amount of ZEN plus α -ZEL (average per group; n 3) was determined on an Agilent 1290 series UHPLC system connected to a 6500QTrap mass spectrometer in each group of urine samples control group was fed ZEN containing food but NO ZEN degrading polypeptide SEQ ID NO:1, variant a and variant B groups were fed the same food as control group but additionally contained 2.5U/kg food, 5U/kg food, 10U/kg food or the designated ZEN degrading polypeptide of 20U/kg food.
Figure 7 analysis results of stool samples from pig feeding trials compared to SEQ ID NO:1 total amount of ZEN plus α -ZEL per gram of freeze dried stool (average per group; n 3) was determined on Agilent 1290 series UHPLC system connected to 6500QTrap mass spectrometer control group was fed ZEN containing food but NO ZEN degrading polypeptide group SEQ ID NO:1 group, variant a group and variant B group were fed the same food as control group but additionally contained 2.5U/kg food, 5U/kg food, 10U/kg food or the designated ZEN degrading polypeptide of 20U/kg food, change in amount of ZEN plus α -ZEL in stool compared to SEQ ID NO:1 is shown in percentage.
FIG. 8: 6500 selected response monitoring parameters on QTrap were used to analyze samples from the broiler feeding trial. Analysis of samples from the chick feeding trial was performed on an Agilent 1290 series UHPLC system connected to a 6500QTrap mass spectrometer. Selected reaction monitoring parameters are shown. The product ions are given quantitatively/qualitatively.
FIG. 9: analysis results of crop samples from the chick feeding trial compared to SEQ ID NO: 1. The concentration of ZEN per kg of freeze-dried crop sample (mean value per group; n-8) was determined on an Agilent 1290 series UHPLC system connected to a 6500QTrap mass spectrometer. Control groups were fed ZEN-containing food, but no ZEN-degrading polypeptides. The other groups were fed the same food as the control group, but additionally contained the specified amount of enzymatically active ZEN-degrading polypeptide variant B. The change in concentration of ZEN in crop compared to the control is shown as a percentage.
Detailed Description
It has surprisingly been found that α/β -hydrolases comprising the mutations described herein in specific regions, i.e., the VI-domain and the CAP-loop, exhibit greater temperature stability and/or pH stability.
The inventors recognized the CAP-domain of SEQ ID NO:1 as amino acids at positions 145 to 218, the CAP-domain of SEQ ID NO:2 as position 144-218, the CAP-domains of SEQ ID NO:3, 4 and 5 as position 145-218, and the CAP-domain of SEQ ID NO:6 as position 161-235. Furthermore, the inventors of the present application recognized the VI-domain of SEQ ID NO:1 as
Mutations as described herein that introduce a CAP-domain, in particular a VI-domain as defined herein, or more specifically a CAP-loop as defined herein, provide sufficient temperature stability without loss of the active properties and/or pH stability, such that such enzymes can be used in processes at elevated temperatures.
This is particularly important because in the food and feed industry, heat treatments such as pelletization are often used to produce hygiene products with reduced microbial loads.
Pelleting of feed is a particularly common standardized method to enhance flowability, reduce dust formation and reduce microbial (especially salmonella) load. During granulation, the product is usually wetted by hot steam, heated and then compressed under pressure through a matrix (matrix). Such heat treatment of an enzyme or polypeptide often results in a reduction of its enzymatic activity and/or its irreversible denaturation.
Furthermore, when the enzyme is used in food or feed, it is typically subjected to inactivation by conditions within the gastrointestinal tract of the animal. In particular, a low pH environment may result in a temporary or permanent reduction or even elimination of the enzymatic activity of the enzyme or polypeptide.
However, enzymes that degrade ZEN typically have low temperature stability and/or pH stability and thus cannot be incorporated into feed or food. Thus, the use of polypeptides or enzymes as additives for the pelletization of food or feed presents considerable technical challenges.
The α/β -hydrolases described herein have increased stability, especially in terms of temperature and/or pH stability, and are therefore very suitable for use in food and feed production processes.
Thus, the present invention relates to a method for increasing the stability of α/β -hydrolase, said α/β -hydrolase comprising a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, or a sequence having 58% or more sequence identity to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1 (CAP-domain; 58% identity to CAP-domain of SEQ ID NO: 1), said method comprising:
replacing at least one amino acid at the following positions:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein the amino acid is replaced with an amino acid that: the amino acids used have a more negative hydrophilicity index than the amino acids being replaced,
wherein the hydropathic index is determined by Kyte and Doolittle hydropathic index,
thereby an α/β -hydrolase with increased stability is obtained, preferably said α/β -hydrolase has increased stability compared to the α/β -hydrolase before the amino acid substitution and/or said α/β -hydrolase has increased stability compared to the α/β -hydrolase without said amino acid substitution.
The present invention also relates to a method for increasing the stability of α/β -hydrolase, said α/β -hydrolase comprising a sequence corresponding to positions 161 to 235 of SEQ ID No. 6, or a sequence having 58% or more sequence identity to a sequence corresponding to positions 161 to 235 of SEQ ID No. 6 (CAP-domain; 58% identity to CAP-domain of SEQ ID No. 6), said method comprising:
replacing at least one amino acid at the following positions:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein the amino acid is replaced with an amino acid that: the amino acids used have a more negative hydrophilicity index than the amino acids being replaced,
wherein the hydropathic index is determined by Kyte and Doolittle hydropathic index,
thereby an α/β -hydrolase with increased stability is obtained, preferably said α/β -hydrolase has increased stability compared to the α/β -hydrolase before the amino acid substitution and/or said α/β -hydrolase has increased stability compared to the α/β -hydrolase without said amino acid substitution.
Increased stability as used herein may mean that the α/β -hydrolase of the invention has a higher stability compared to the α/β -hydrolase comprising a sequence corresponding to positions 145 to 218 of SEQ ID NO:1 (and SEQ ID NOs: 3,4, 5). alternatively or additionally, increased stability as used herein means that the α/β -hydrolase of the invention has a higher stability compared to the α/β -hydrolase comprising a sequence corresponding to positions 144 to 217 of SEQ ID NO: 2. alternatively or additionally, the α/β -hydrolase comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO: 6.
Increased stability as used herein may also mean a higher stability of the β 0/β 1-hydrolase of the invention compared to the α/β -hydrolase of SEQ ID NO:1 alternatively or additionally, increased stability as used herein may also mean a higher stability of the α/β -hydrolase of the invention compared to the α/β -hydrolase of SEQ ID NO:2 alternatively or additionally increased stability as used herein may also mean a higher stability of the α/β -hydrolase of the invention compared to the α/β -hydrolase of SEQ ID NO: 6.
This includes, for example, β 0/β 1-hydrolase of increased stability obtained by the methods of the invention or α/β -hydrolase of the invention having increased stability compared to α/β -hydrolase without the substitutions described herein, similarly, α/β -hydrolase of increased stability obtained by the methods of the invention or α/β -hydrolase of the invention has increased stability compared to α/β -hydrolase before the amino acids disclosed herein are substituted.
The skilled artisan is aware of various α/β -hydrolases, described in particular in Lenfant et al, (2013) 'ESTHER, The database of The β/β -hydrolases fold surficial of proteins: tools of The genomic nature of functions' Nucleic Acids Research, Volume 41, Issue D1, D423-D429 and Mindrebo et al, (2016) 'uneiling The functional nature of The Alpha-Beta hydrolases fold plants' Curr Opin Oput 233-246. all
In most family members, the β -strands are in a parallel orientation, but some members have a first strand inversion, resulting in a trans-parallel orientation the prototype of the enzyme in the fold has a catalytic triad consisting of a nucleophilic residue at the top of the gamma-turn between the fifth β -strand and the following α -helix (the nucleophile elbow), an acidic amino acid residue (glutamic acid or aspartic acid) and a histidine residue.
α/β -members of different classes of hydrolases and their structural features are described in particular in Kourist et al, (2010) 'The alpha/beta-hydrosase fold 3DM database (ABHDB) as a tool for protein engineering.' Chemobiochem.11 (12): 1635-43).
As enzymes, α/β -hydrolase is generally described as being responsible for the hydrolysis of ester and peptide bonds, however, α/β -hydrolase also participates in the cleavage of carbon-carbon bonds, decarboxylation, and co-factor-free (cofactor-independent) double oxidation of heteroaromatic rings α/β -hydrolases may include catalytic members (enzymes) in this superfamily 2O 2Or O 2And not H 2Activation of O is an enzyme used in the reaction mechanism (haloalkane dehalogenase, haloperoxidase, hydroxynitrile lyase). Non-catalytic members may include neurotropin, gluten (glutamin), neurochemokines, the C-terminal domain of thyroglobulin, vitellin, CCG 1-interacting factor-B and dipeptidyl aminopeptidase VI.
Thus, one skilled in the art can also obtain α/β -hydrolase from ESTHER (a database of α/β -hydrolase-folded protein superfamily).
One skilled in the art can also determine α/β -hydrolase contains a CAP-domain.
1. The α/β -hydrolase was retrieved in an online enzyme database or by comparing a given sequence to SEQ ID NOS: 1-6.
2. It is preferably determined α/β -hydrolase whether it contains a CAP-domain, VI-domain or CAP-loop by using the procedure described in example 2.
3. It is preferably determined whether α/β -hydrolase containing a CAP-domain, VI-domain or CAP-ring can hydrolyze ZEN by employing the procedure described in example 4.
The α/β -hydrolase as described herein comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, or a sequence (CAP-domain) having 58% or more sequence identity to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1 thus, any α/β -hydrolase comprising this sequence is encompassed within the term α/β -hydrolase this sequence corresponds to the CAP-domain of the α/β -hydrolase of SEQ ID NO: 1.
Additionally or alternatively, a α/β -hydrolase as described herein may further comprise a sequence corresponding to positions 144 to 217 of SEQ ID NO:2, or a sequence (CAP-domain) having 58% or more sequence identity to a sequence corresponding to positions 144 to 217 of SEQ ID NO: 2.
Additionally or alternatively, a α/β -hydrolase as described herein may further comprise a sequence corresponding to positions 145 to 218 of
Additionally or alternatively, a α/β -hydrolase as described herein may further comprise a sequence corresponding to positions 161 to 235 of SEQ ID No. 6, or a sequence (CAP-domain) having 58% or more sequence identity to a sequence corresponding to positions 161 to 235 of SEQ ID No. 6.
For example, the α/β -hydrolase may comprise a sequence that is at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identical to the sequence of SEQ ID NO. 1. additionally or alternatively, the α/β -hydrolase may comprise a sequence that is at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identical to the sequence of SEQ ID NO. 2. additionally or alternatively, the α/β -hydrolase may comprise a sequence that is at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identical to the sequence of SEQ ID NO. 3,4, and/or 5. additionally or alternatively, the α/β -hydrolase may comprise a sequence that is at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 97%, 99%, 98%, 99% identical to the sequence of SEQ ID NO. 6.
As used herein, the term "polypeptide" means a peptide, protein or polypeptide, which may be used interchangeably and encompasses an amino acid chain of a given length in which the amino acid residues are linked by covalent peptide bonds. Amino acids other than the 20 proteinogenic amino acids of the standard genetic code known to those skilled in the art, such as selenocysteine, are also encompassed by the present invention. Such polypeptides include any of SEQ ID NOs 1-6.
The term polypeptide also refers to, and does not exclude, modifications of the polypeptide. Modifications include glycosylation, acetylation, acylation, phosphorylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation (formulation), gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation (selenoylation), sulfation, transfer-RNA mediated addition of amino acids such as arginylation and ubiquitination to proteins; see, e.g., PROTECTINS-STRUCTURE AND MOLECULAR PROPERTIES,2nd Ed., T.E.Creighton, W.H.Freeman AND company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B.C. Johnson, Ed., Academic Press, New York (1983), pgs.1-12; seifter, meth.enzymol.182 (1990); 626 + 646, Rattan, Ann.NY Acad.Sci.663 (1992); 48-62.
According to the present invention, the term "identical" or "percent identity" in the context of two or more polypeptide sequences (such as SEQ ID NOs: 1-6) refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) that are the same, when compared and aligned for maximum correspondence over a comparison window or over a designated region of measurement using sequence comparison algorithms known in the art or by manual alignment and visual inspection. Sequences having, for example, 80% to 95% or more sequence identity are considered to be substantially identical. This definition also applies to the complement of the test sequence. The person skilled in the art will know how to determine the percentage identity between/in sequences using, for example, those based on the CLUSTALW computer program (Thompson Nucl. acids Res.2(1994), 4673-.
Also available to those skilled in the art are the BLAST and BLAST2.6 algorithms (Altschul Nucl. acids sRs.25 (1977), 3389-3402). The BLASTP program for amino acid sequences uses the default word size (W)6, the
For example, BLAST2.6(Altschul, Nucl. acids SRes.25(1997), 3389-.
As used herein, "CAP-domain" refers to The CAP-domain of α/β -Hydrolase, as described for example in Kourist et al, (2010) ' The alpha/beta-hydrosilase Fold 3DM database (ABHDB) as a topol for Protein engineering. ' Chemiochem.11 (12):1635-43 in FIG. 1 or in Carr and Ollis (2009) ' β/β 0 hydrosilase Fold: An update. ' Protein & Peptide Letters,2009,16(10): 1137-1148. it is also envisaged that The CAP-domain may be located between β -Fold and β -3-helices, for example between b6 and aD of α/β -Hydrolase, for example Ollis et al, (1992) ' The beta-hydrosilase Fold 3-helices, and may be located after The start domain of CAP-domain, for example in The end of The CAP-domain, and/The fragment 76-domain may be located after The start domain of The CAP-linker, for example in The CAP-domain of The CAP-19-linker, for example, The CAP-domain of The CAP-domain, and The end domain of The CAP domain, for example, The CAP-domain of The CAP, The CAP-.
The process of the invention requires the replacement of at least one amino acid at the following positions of α/β -hydrolase:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
As used herein, a 'VI-domain' is a portion of a CAP-domain. Thus, the CAP-domain comprises a VI-domain. The VI-domain can start at the first amino acid after the QXAGP motif (SEQ ID NO:7) present in the CAP-domain and can continue to the last amino acid before the EYDPE motif (SEQ ID NO:8), wherein the EYDPE motif is not part of the VI-domain. These motifs are underlined in the sequences described in table 2 herein.
For example, the VI-domain may comprise a sequence corresponding to
The position of a given amino acid that may be substituted according to the present invention may be changed due to a deleted or added amino acid, or the position of a given amino acid that may be substituted may be changed due to a deletion or addition of an amino acid elsewhere in the (mutant or wild-type) α/β -hydrolase.
Thus, by "corresponding positions" according to the invention, it is preferably understood that the amino acids may differ in the indicated numbering, but may still have similar adjacent amino acids. The term "corresponding position" also includes said amino acids which may be exchanged, deleted or added. In particular, when aligning, for example, any of SEQ ID NOs:1-6, preferably the reference sequence of SEQ ID NO:1 (subject sequence) with the amino acid sequence of interest (query sequence), one skilled in the art can, for example, examine the sequence of interest in finding the amino acid positions described herein (i.e., positions corresponding to positions 185 and/or 188 of the amino acid sequence shown in SEQ ID NO: 1) to find the sequence of SEQ ID NO:1 (or the corresponding amino acid sequence encoding the protein).
In the method of the present invention, the amino acid is replaced with an amino acid that: the amino acids used have a hydropathic index that is more negative than the amino acids being replaced, wherein the hydropathic index is determined by the Kyte and Doolittle hydropathic indices.
As used herein, "amino acid substitution" means the substitution of an amino acid relative to the corresponding position of the identified SEQ ID NO (e.g., any one of the positions specified herein of SEQ ID NO: 1-6). For example, in one embodiment, the substitution is an amino acid substitution relative to the amino acid at a position corresponding to
The "hydrophilicity index," also referred to herein as the "hydrophilicity value," is a number representing the hydrophobicity or hydrophilicity of an amino acid side chain. In particular, for hydropathic index, each amino acid has been assigned a value reflecting its relative hydrophilicity. Thus, the hydrophilicity of an amino acid can be determined by the hydropathic index. Jack Kyte and Russell F.Doolittle (Kyte and Doolittle (1983) 'A simple method for displaying the hydropathic character of epitope' J.mol.biol.157(1): 105-32) propose this hydropathic index of amino acids. The amino acids with the least negative hydropathic index are isoleucine (4.5) and valine (4.2). According to Kyte and Doolittle, the amino acids with the most negative hydropathic indices are arginine (-4.5) and lysine (-3.9). Hydropathic index is considered important in protein structure. Amino acids with a less negative hydropathic index tend to be internal (in terms of the three-dimensional shape of the protein) while amino acids with a more negative hydropathic index are more common on the surface of the protein. The hydropathic indices of Kyte and Doolittle have been summarized in table 1 herein:
table 1: hydropathic indices of Kyte and Doolittle
It is further contemplated that at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more amino acids are substituted.
The method also contemplates replacing at least one amino acid at the following positions:
positions corresponding to positions 185 to 191 of SEQ ID NO.1, and/or
Positions corresponding to positions 184 to 190 of
Positions corresponding to positions 185 to 191 of
Positions corresponding to positions 201 to 208 of
All these positions are located within the CAP-ring.
In this context, it is noted that the CAP-domain and the VI-domain may further comprise loops (sequences/domains). This 'loop', also referred to herein as a 'CAP-loop', can start after the first amino acid after the G (F/Y) XXAA (SEQ ID NO:9) motif present in the VI-domain and can continue until the last amino acid before the ARXF motif (SEQ ID NO:10) (or the QLFP motif for SEQ ID NO:6 (SEQ ID NO:11)), where the ARXF motif (SEQ ID NO:10) (or the QLFP motif for SEQ ID NO:6 (SEQ ID NO:11)) is not part of the CAP-loop. These motifs are underlined in the sequences described in table 2 below.
For example, the CAP-loop may comprise a sequence corresponding to positions 185 to 191 of SEQ ID No.1, or a sequence having at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to a sequence corresponding to positions 185 to 191 of SEQ ID No. 1. Additionally or alternatively, the CAP-loop may comprise a sequence corresponding to positions 184 to 190 of SEQ ID No. 2, or a sequence having at least 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to a sequence corresponding to positions 184 to 190 of SEQ ID No. 2. Additionally or alternatively, the CAP-loop may comprise a sequence corresponding to positions 185 to 191 of
Thus, an α/β -hydrolase as described herein may comprise a VI-domain and/or a CAP-ring as described herein.
It is also contemplated to replace the amino acid with an amino acid selected from R, K, N, Q, D, E, H, P, Y, W, S, T, G, A, M, C, F, L or V.
It is further contemplated to replace the amino acid with an amino acid selected from R, K, N, Q, D, E, H, P, Y, W, S, T or G.
It is also contemplated to replace the amino acid with an amino acid selected from R, K, N, Q, D, E, H or P.
It is further contemplated to replace the amino acid with an amino acid selected from R, K, D, Q, D, N, E, P, G, T, S or H.
It is also contemplated to replace the amino acid with an amino acid selected from S, P, R, D, H, G or N. The amino acid may also be replaced with an amino acid selected from R, D, H, G or N.
It is also contemplated to replace the amino acid with an amino acid selected from P, S, R or H. The amino acid may also be replaced with an amino acid selected from R or N.
It is further contemplated that the amino acid substitution is selected from one or more of: v → A, G → R, G → S, A → P, A → R, A → D, A → H, A → N, A → G, S → D, P → H, M → D, G → E, I → A, I → V, H → N, Q → K, F → Y and/or V → C.
The amino acid substitutions may also be selected from one or more of the following: V160A, G185R, G185S, a186P, a186R, a188D, a188H, a188N, a188G, a188R, S189D, P190H, M191D, G199E, I200A, I200V, H203N, Q205K, F183Y and/or V197C.
It is also contemplated that the amino acid substitution is selected from one or more of the following: v → A, G → R, G → S, A → P, A → R, A → D, A → H, A → N, A → G, S → D, P → H, M → D, G → E, I → A, I → V, H → N and/or Q → K. The amino acid substitutions may also be selected from one or more of the following: V160A, G185R, G185S, a186P, a186R, a188D, a188H, a188N, a188G, a188R, S189D, P190H, M191D, G199E, I200A, I200V, H203N, and/or Q205K.
The amino acid substitutions may also be selected from G185R, a186R, a188R, a188D, a188H, a188N and/or M191D.
It is further contemplated that the amino acid is replaced with an amino acid selected from R, D, H, G, N or P.
It is also contemplated that the methods of the invention comprise substituting at least one amino acid at the following positions:
positions corresponding to positions 185 to 191 of SEQ ID NO.1, and/or
Positions corresponding to positions 184 to 190 of
Positions corresponding to positions 185 to 191 of
Positions corresponding to positions 201 to 208 of
And wherein the amino acid is replaced with an amino acid selected from R, D, H, G, N or P.
The present invention relates to a method for increasing the stability of α/β -hydrolase, which may be a decrease in GRAVY value, an increase in pH stability and/or an increase in temperature stability.
As used herein, the "GRAVY value" of a protein is a measure of its relative hydrophobicity or hydrophilicity. The two measures are combined in a hydropathic scale or hydropathic index. The GRAVY values are calculated according to Kyte and Doolittle (Kyte J, Doolittle RF (May1983). "A simple method for displaying the hydropathic graph of aprotein". J.Mol.biol.157(1): 105-32) by adding the hydropathic value (hydropathic index, see Table 1 above) of each residue and dividing by the number of residues in the sequence. Thus, the GRAVY value can be calculated by dividing the sum of the hydropathic values (indices) of all amino acids by the number of amino acid residues in the sequence (calculated according to Kyte and Doolittle). As used herein, the term "temperature stability" refers to the property of an enzyme to maintain its catalytic activity after temporary exposure to elevated temperatures. The temperature stability is determined by measuring and comparing the enzyme activity of an enzyme or polypeptide solution before and after a 10 minute heat treatment or without heat treatment under the same defined conditions.
In particular, the temperature stability can be measured in the following manner. The polypeptides were diluted to a concentration of 0.001526923U/ml with sample buffer (Teorell Stenhagen buffer pH7.5 (Stenhagen & Teorell. (1938) Nature 141,415), containing 0.1mg/ml bovine serum albumin) and placed on ice until use. 40 aliquots of 50. mu.l of the diluted polypeptide solution were transferred to 4 sets of 12-linked tubes (e.g., from tarlab) with the first and last tube of each set of linked tubes empty. The tubing was sealed with a 12-piece cap (e.g., from starlab). As a positive control, 4 aliquots of 50. mu.l of diluted enzyme solution were transferred to 4 PCR tubes. All PCR tubes and connecting tubes were placed on ice until the temperature incubation step was initiated. As a negative control, 4 aliquots of 50 μ l sample buffer were transferred to 4 PCR tubes. These tubes were stored at 25 ℃.
ZEN degradation assay buffer (Teorell Stenhagen buffer, pH7.5, containing 0.1mg/ml bovine serum albumin and 5.3ppm ZEN) was prepared and an aliquot of 660. mu.l of assay buffer was transferred to 48 reaction tubes. The tubes were sealed and stored at 25 ℃ until the ZEN degradation analysis started. For degradation analysis, 40 μ l of each of the 40 temperature treated samples from the PCR run, 40 μ l of each of the 4 negative controls and 40 μ l of each of the 4 positive controls were added to the tube containing 660 μ l of assay buffer, thereby achieving a final ZEN concentration of 5ppm in the assay reaction. Furthermore, the final concentration of polypeptide was also achieved thereby to effectively degrade ZEN within three hours (i.e. 90% -100% ZEN degradation).
Degradation analysis was initiated by adding temperature treated samples, positive or negative controls to the assay buffer. The ZEN degradation reaction was incubated in a preheated water bath at 37 ℃. Immediately after the start of the degradation reaction, mixing by vortex was carried out for about 2 seconds, and 100. mu.l of the 0h sample was transferred to a new reaction tube. After 0.5, 1.0, 2.0 and 3.0 hours, additional samples were taken from the ZEN degradation analysis reaction. Once the sample is extracted from the degradation reaction, the enzyme in the sample is heat inactivated by incubation at 99 ℃ for 10 minutes. Subsequently, the tubes were centrifuged (2 min, 25 ℃, 14674xg) and 90 μ Ι of supernatant was transferred to HPLC vials with inserts. These HPLC vials were stored at 4 ℃ until HPLC-DAD measurements were performed as described in example 4.
Enzyme activity was calculated in units per liter (U/l) using a linear decrease in ZEN concentration as determined by HPLC-DAD analysis of ZEN degraded samples. One unit is defined as the amount of enzyme activity that degrades 1 μmol ZEN in 1 minute under the conditions described. The residual activity after incubation for 0, 5, 10 and 20 minutes at different temperatures was calculated as follows: the enzyme activity in the temperature treated samples was divided by the average value of the enzyme activity of the positive controls and multiplied by 100.
The temperature stability (T (50%)) is defined as the temperature: the polypeptide after 10 minutes incubation at this temperature had 50% residual activity compared to the positive control. The following examples are given for illustration: the parent enzyme had an enzyme activity of 50U/ml after 10 minutes incubation on ice and 25U/ml after 10 minutes incubation at 59.3 ℃ and a T (50%) value of 59.3 ℃. If the enzyme variant has a T (50%) value of 61.0 ℃, the relative increase in temperature stability (T (50%)) compared to the parent enzyme is 2.9%. This is obtained by dividing the difference between the two T (50%) values by 1.7 ℃ by the T (50%) value of the parent enzyme of 59.3 ℃ and multiplying by 100.
Thus, temperature stability as used herein is a measure of the resistance of an enzyme activity to inactivation when temporarily exposed to a temperature selected from 20 ℃ to 85 ℃. The temperature at which the residual activity of the heat-treated enzyme after 10 minutes of incubation was 50% can be compared with a positive control. An increase in T (50%) of a polypeptide variant relative to its parent polypeptide is defined herein as increased temperature stability, and such an increase may be expressed relatively as a percentage value or absolutely in degrees celsius.
The term "pH stability" as used herein refers to the property of a polypeptide to maintain its catalytic activity after a temporary incubation at a specific pH, and thus "pH stability" is reflected by the residual activity of the polypeptide after the temporary incubation at a specific pH. The residual activity after incubation at a specific pH was determined by comparing the enzymatic activity of the polypeptide solution to degrade ZEN after incubation for 60 minutes in buffers of different pH with the enzymatic activity of the same polypeptide solution after incubation for 60 minutes in buffer at pH 7.5. The pH stability is a measure of the resistance of an enzyme to temporary exposure to a specific pH environment. An increase in pH stability is defined as an increase in residual activity of the polypeptide variant after incubation at pH4.0 (═ pH treatment) compared to the residual activity of the parent enzyme variant after incubation at pH 4.0.
The pH stability can be measured as follows. ZEN-degrading polypeptides were incubated in buffer solutions at various pH values for 1 hour. Aliquots containing the polypeptide variants were transferred to 8 sample tubes containing 8 incubation buffers at different pH values. The incubation buffer is a milk-free and semi-concentrated Fed State complexed fluidic intermediate buffer (Jantrat et al, (2008) 'separation medium interactions in the proto-fluidic product: an update' Pharm Res.2008 Jul; 25(7): 1663-76). The pH of the incubation buffer in the 8 sample tubes was set to 3.5, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 and 6.0, respectively. Also 1 aliquot of the polypeptide variant was transferred to a tube containing sample buffer (Teorell Stenhagen buffer, pH7.5, containing 0.1mg/ml bovine serum albumin) as a positive control. As a negative control, 100. mu.l of sample buffer was incubated in a pre-heated water bath at 37 ℃ for 1 hour. After incubation, the ability of the sample to degrade ZEN was determined in an assay buffer solution similar to that described elsewhere herein or in the examples (e.g., example 4). The addition of ZEN degradation assay buffer ensured that the pH was constant at pH7.5 in all samples. Samples were taken throughout the ZEN degradation analysis reaction and analyzed for ZEN, Hydrolyzed Zearalenone (HZEN) and Decarboxylated Hydrolyzed Zearalenone (DHZEN) concentrations using HPLC-DAD measurements as described in example 4, for example. The activity is calculated, for example, as described in example 4.
An increase in pH stability is defined as an increase in residual activity of the polypeptide solution after incubation at pH4.0 compared to the residual activity of the unmutated parent enzyme solution after the same treatment. The residual activity is defined by comparing the activity of the pH-treated polypeptide solution with the activity of the same polypeptide variant solution after incubation at pH 7.5. The residual activity was calculated as follows: the enzyme activity of the pH-treated sample was divided by the enzyme activity of the control incubated at pH7.5 and multiplied by 100. The following examples are given for illustration: if the enzyme activity of a polypeptide sample after incubation at pH4.0 is 0.5U/l and the enzyme activity of the polypeptide after incubation at pH7.5 is 2.7U/l, the residual activity is 18.5%. If the residual activity of the parent polypeptide SEQ ID NO:1 after incubation at pH4.0 is measured to be 2.5%, the pH stability of the polypeptide variant is increased by a factor of 7.4 compared to the parent polypeptide.
The present invention also relates to an α/β -hydrolase obtainable by the process described herein.
The invention also relates to an α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO:1 or a sequence having more than 58% sequence identity to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1,
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein said α/β -hydrolase has a GRAVY value that is at least 0.6% negative compared to the GRAVY value of a α/β -hydrolase having the polypeptide sequence of SEQ ID NO: 1.
The invention also relates to an α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO. 6 or a sequence having more than 58% sequence identity to a sequence corresponding to positions 161 to 235 of SEQ ID NO. 6,
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein said α/β -hydrolase has a GRAVY value that is at least 0.6% negative compared to the GRAVY value of a α/β -hydrolase having the polypeptide sequence of
It is also expected that the α/β -hydrolase has a GRAVY value that is at least 3.0%, 4.2%, 4.8%, 6.0%, 6.6%, 7.8%, 10.2%, 12.0% or higher than the GRAVY value of a α/β -hydrolase having the polypeptide sequence of SEQ ID NO: 1.
It is also expected that the α/β -hydrolase has a GRAVY value that is at least 1.0%, 2.0%, 2.5%, 2.6%, 3.0%, 4.0%, 5.0%, 6.0%, 6.8% or higher lower compared to the GRAVY value of a α/β -hydrolase having the polypeptide sequence of SEQ ID No. 6.
The invention also relates to an α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO:1 or a sequence having more than 58% sequence identity to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1,
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 184 to 190 of
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 201 to 208 of
wherein the amino acid substitution is selected from V → A, G → R, G → S, A → P, A → R, A → D, A → H, A → N, A → G, S → D, P → H, M → D, G → E, I → A, I → V, H → N and Q → K, and/or
Wherein an amino acid selected from P, R, D, H, G or N is substituted for the amino acid, preferably the amino acid employed is selected from R, D, H, G or N, more preferably the amino acid employed is selected from R or N such α/β -hydrolase may have greater stability than the same α/β -hydrolase without such substitution or prior to the introduction of such substitutions, e.g., such α/β -hydrolase may have greater stability than the α/β -hydrolase of SEQ ID NO: 1.
The invention also relates to an α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO. 6 or a sequence having more than 58% sequence identity to a sequence corresponding to positions 161 to 235 of SEQ ID NO. 6,
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 184 to 190 of
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 201 to 208 of
wherein the amino acid substitution is selected from F → Y or V → C. such α/β -hydrolases may have greater stability than the same α/β -hydrolase without such substitution or prior to the introduction of such substitutions, for example, such α/β -hydrolases may have greater stability than the α/β -hydrolase of SEQ ID NO. 6.
It is contemplated that the α/β -hydrolase as described herein comprises the following amino acid substitutions:
-G → R and a → N, preferably G185R and a 188N;
-G → S and a → R, preferably G185S and a 188R;
-G → R, A → R, A → H, S → D, P → H and M → D, preferably G185R, a186R, a188H, S189D, P190H and M191D;
-V → A, G → R, A → N, G → E, I → A, H → N and Q → K, preferably V160A, G185R, a188N, G199E, I200A, H203N and Q205K;
-V → A, G → S, A → R, G → E, I → A, H → N and Q → K, preferably V160A, G185S, a188R, G199E, I200A, H203N and Q205K;
-V → A, G → E, I → A, H → N and Q → K, preferably V160A, G199E, I200A, H203N and Q205K;
-V → A, G → R, A → R, A → H, G → E, I → V, H → N and Q → K, preferably V160A, G185R, a186R, a188H, G199E, I200V, H203N and Q205K; and/or
F → Y and V → C, preferably F183Y and V197C.
The present invention also relates to nucleic acid molecules encoding the α/β -hydrolases described herein, which may be introduced or inserted into an expression vector the term "expression vector" refers to a nucleic acid molecule construct capable of expressing a gene in vivo or in vitro.
The expression vectors described herein may be expressed in a host cell. The term "host cell" refers to all such cells: which comprises the nucleotide sequence to be expressed or comprises an expression vector and is capable of producing the enzyme or polypeptide according to the invention. In particular, the term refers to prokaryotic and/or eukaryotic cells, preferably to spores of pichia pastoris (pichia pastoris), Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Streptomyces (Streptomyces), Hansenula (Hansenula), Trichoderma (Trichoderma), Lactobacillus (Lactobacillus), Aspergillus (Aspergillus), plant cells and/or Bacillus (Bacillus), Trichoderma (Trichoderma) or Aspergillus (Aspergillus). The designation Pichia pastoris (P.pastoris) as used herein has The same meaning as The designation Komagataella pastoris, where Pichia pastoris is an older designation and K.pastoris is a newer designation of The system (Yamada et al, (1995) 'The genetic relationships of methane-organizing Yeast Based on The Partial sequence of 18S and 26S Ribosom RNAs: The Proposal of Komagataella Gen. nov. (Saccharomyces cerevisiae)' Bioscience, Biotechnology Biochemistry, Vol.59, issue3, pp.439-444). Notably, the species of Komagataella pastoris has recently been reassigned to Komagataella phaffii (Kurtzman (2009) "Biotechnology strains of Komagataella (Pichia) as determined from multigenetic analysis." J Ind Microbiol Biotechnology.36 (11): 1435-8). The Komagataella phaffii used herein may for example relate to the strain Komagataella phaffii CBS 7435, Komagataella phaffii GS115 or Komagataella phaffii JC 308.
The present invention also relates to the use of the α/β -hydrolase described herein for the degradation of Zearalenone (ZEN).
ZEN is a non-steroidal estrogenic macrolide having the following structural formula, which is synthesized by the polyketide metabolic pathway:
and, according to the IUPAC nomenclature, its name is (2E,11S) -15, 17-dihydroxy-11-methyl-12-oxabicyclo [12.4.0] octadecane-1 (18),2,14, 16-tetraene-7, 13-dione.
However, there are also many ZEN derivatives in nature, which can be formed by enzymatic or chemical modification of ZEN, examples include glycoside ZEN conjugates formed by metabolism of fungi, plants or mammals or those containing sulfates, and ZEN metabolites formed in the human or animal body, and so on, ZEN derivatives are understood as ZEN conjugates or ZEN metabolites naturally occurring or synthesized by chemical or biochemical synthesis, but specifically refer to α -zearalenol (α -ZEL; (2E,7R,11S) -7,15, 17-trihydroxy-11-methyl-12-oxabicyclo [12.4.0] octadeca-1 (18); 2,14, 16-tetraene-13-one; (2E,7R,11S) -7,15, 17-trihydroxy-11-methyl-11-oxabicyclo [12.4.0] octadecane-1 (18); (2E,7S,11S) -7,15, 17-11-methyl-12-oxabicyclo [12.4.0] octadecane-1, 2,14, 16-tetraene-13-one; (β -ZEL; (2E,7S,11S) -7,15, 17-methyl-12-oxabicyclo [12.4.0] octadecane-12, 15-oxa-12-hydroxy-2, 15-oxa-5-oxa-oxabicyclo [ 11-13 ] ketone (11-12, 15-5-oxa-5-oxa-12-oxa-11-17-11-12-oxa-17-6-hydroxy-oxa-2, 15-6-oxa-oxabicyclo [ 11-oxa-17-oxa-methyl-17-11-17-15-methyl-oxa-17-11-17-12-oxa-methyl-oxa-11-12-17-oxa-11-12-hydroxy-oxa-17-12-15-17-15-oxa-15-17-oxa-17-15-oxa.
Due to its high chemical and physical stability ZEN and ZEN derivatives can also be detected in processed food and animal feed products such as bread or beer, especially α -ZEL, β -ZEL, Z14S, α -ZAL, β -ZAL, Z14G and ZAN.
ZEN and ZEN derivatives were successfully hydrolyzed using any one of the polypeptides having sequence ID numbers
the hydrolysis of ZEN to form non-toxic Hydrolyzed Zearalenone (HZEN) and/or hydrolyzed ZEN derivatives is carried out by the α/β -hydrolase of the present invention it is believed that further decarboxylation of HZEN to decarboxylated hydrolyzed ZEN (dhzen) and/or decarboxylated hydrolyzed ZEN derivatives occurs spontaneously.
The α/β -hydrolase described herein is capable and suitable for degrading ZEN, for example, the α/β -hydrolase can be adapted to hydrolytically cleave the ester group of ZEN and/or its derivatives independent of oxygen and without co-factors.
ZEN degradation can be measured by adding α/β -hydrolase of the invention to Teorell Stenhagen buffer (pH 7.5) with 0.1mg/ml bovine serum albumin at a temperature of 37 ℃.
Specifically, ZEN degradation can be measured as follows in a sample buffer containing 0.1mg/ml bovine serum albumin (Teorell Stenhagen buffer (Stenhagen & Teorell. (1938) Nature 141,415) at pH7.5 at a temperature of 37 ℃ for 3 hours, polypeptide/enzyme is diluted with the sample buffer and placed on ice for use, as a negative control, the sample buffer containing 5 μ g/ml ZEN is incubated, for the degradation group (appaach), the sample buffer containing 5 μ g/ml ZEN is mixed with the polypeptide/enzyme solution to reach a final enzyme concentration that degrades the available ZEN to an extent of 90% to 100% within 3 hours, the reaction is started by adding polypeptide or enzyme to the degradation group, no enzyme is added to the negative control, vortex for about 2 seconds immediately after the start of the respective reaction, and the 0h sample (100 μ l) is transferred to a new reaction tube, the water bath of the reaction is incubated at 37 ℃, the samples were heat-inactivated by incubation at 99 ℃ for 10min, centrifuged (2 min, 25 ℃, 14674xg) and 90 μ Ι of supernatant was transferred to HPLC vials with inserts. The samples were stored at 4 ℃ until HPLC-DAD measurement. Samples were taken repeatedly after 0.5, 1.0, 2.0 and 3.0 hours.
The concentrations of ZEN, HZEN and DHZEN can be analyzed by HPLC-DAD as described in Vekiru et al (Vekiru et al, (2016) 'Isolation and catalysis of enzymatic hydrolysis reaction products' World Mycotoxin Journal 9: 353-. The analysis was performed on an Agilent 1100 series HPLC equipped with a Diode Array Detector (DAD) operating at 274 nm. When the solvent a: 20% aqueous methanol +5mM ammonium acetate and solvent B: 90% aqueous methanol +5mM ammonium acetate, and using the following gradient: 0-0.1min 0% phase B, 0.1-3min increasing linearly to 90% phase B, 3-5min increasing linearly to 100% phase B for 1.9min, then decreasing to 0% phase B within 0.1min, retention time of analyte was 7.03min for ZEN, 5.17min for HZEN and 5.95min for DHZEN when separation was performed at 35 ℃ on Zorbax SB-Aq, 4.6X 150mm, 5 μm column (Agilent Technologies). The column was reconditioned for 2.0min before starting the next run. The flow rate was set to 0.8ml/min and the injection volume was set to 15. mu.l.
Quantification was based on calibration with ZEN, HZEN and DHZEN external standards. Enzyme activity in units of per liter (U/l) was calculated from the slope of the linear range of ZEN degradation determined from a plot of ZEN concentration in the sample versus sampling time points. To determine the amount of enzyme activity in a sample in U/l, the slope of the linear range in the graph described above can be calculated as μ MZEN/hour and divided by 60 to determine μ M/min. By taking into account the possible dilutions and by including these appropriate dilution factors in the calculation, the enzyme activity in the sample can be determined in U/l. The following examples are given for illustration: if the slope of the linear range is 10. mu.M/h, the enzyme activity of the undiluted sample is 0.17U/l; calculated by 10/60 ═ 0.17.
In the present context, it is noted that the term "unit" or "U" refers to a measure of the enzymatic activity and is defined as the amount of micromoles (μmol) of substrate (zearalenone here) reacted or cleaved per minute under defined conditions. The enzyme concentration of an enzyme or polypeptide solution is defined by the "activity" of the enzyme or polypeptide solution, expressed in units per milliliter (U/ml) or units per liter (U/l) of solution.
The present invention also relates to a composition comprising the α/β -hydrolase described herein.
Methods for preparing such food and/or feed compositions are known to the person skilled in the art and are described in particular in WO 99/35240.
The present invention also relates to an α/β -hydrolase or a composition as described herein for use in the treatment or prevention of a disease such as a disease affecting hormonal balance, such as estrogen balance, particularly mycotoxins due to ZEN.
The present invention also relates to a kit comprising the α/β -hydrolase or the composition described herein.
The invention is also characterized by the following items:
1. a method for increasing the stability of α/β -hydrolase, said α/β -hydrolase comprising a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, 3,4,5, and/or comprising a sequence corresponding to positions 144 to 217 of SEQ ID NO:2, and/or comprising a sequence corresponding to positions 161 to 235 of SEQ ID NO:6, and/or comprising a sequence having 58% or more sequence identity to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, 3,4,5, and/or comprising a sequence having 58% or more sequence identity to a sequence corresponding to positions 144 to 217 of SEQ ID NO:2, and/or comprising a sequence having 58% or more sequence identity to a sequence corresponding to positions 161 to 235 of SEQ ID NO:6 (CAP-domain; 58% identity to CAP-domain of SEQ ID NO:1, 2, 3,4,5, 6), said method comprising:
replacing at least one amino acid at the following positions:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein the amino acid is replaced with an amino acid that: the amino acids used have a more negative hydrophilicity index than the amino acids being replaced,
wherein the hydropathic index is determined by Kyte and Doolittle hydropathic index,
α/β -hydrolase having increased stability is thus obtained.
2. The method of
3. the method of
positions corresponding to positions 185 to 191 of SEQ ID NO.1, and/or
Positions corresponding to positions 184 to 190 of
Positions corresponding to positions 185 to 191 of
Positions corresponding to positions 201 to 208 of
4. The method of any one of the preceding items, wherein the amino acid is replaced with an amino acid selected from R, K, D, Q, D, N, E, P, G, T, S or H.
5. The method of any one of the preceding items, wherein the amino acid is replaced with an amino acid selected from R, K, N, Q, D, E, H, P, Y, W, S, T, G, A, M, C, F, L or V.
6. The method of any one of the preceding items, wherein the amino acid is replaced with an amino acid selected from R, K, N, Q, D, E, H, P, Y, W, S, T or G.
7. The method of any one of the preceding items, wherein the amino acid is replaced with an amino acid selected from R, K, N, Q, D, E, H or P.
8. The method of any one of the preceding items, wherein the amino acid is replaced with an amino acid selected from S, P, R, D, H, G or N.
9. The method of any one of the preceding items, wherein the amino acid is replaced with an amino acid selected from R, D, H, G, N or P.
10. The method of any one of the preceding items, wherein the amino acid is replaced with an amino acid selected from R, D, H, G or N.
11. The method of any one of the preceding items, wherein the amino acid is replaced with an amino acid selected from P, S, R or H.
12. The method of any one of the preceding items, wherein the amino acid is replaced with an amino acid selected from R or N.
13. The method of any one of the preceding items, wherein the amino acid substitution is selected from one or more of: v → A, G → R, G → S, A → P, A → R, A → D, A → H, A → N, A → G, S → D, P → H, M → D, G → E, I → A, I → V, H → N and/or Q → K.
14. The method of any one of the preceding items, wherein the amino acid substitution is selected from one or more of: V160A, G185R, G185S, a186P, a186R, a188D, a188H, a188N, a188G, a188R, S189D, P190H, M191D, G199E, I200A, I200V, H203N, and/or Q205K.
15. The method of any one of the preceding items, wherein the amino acid substitution is selected from one or more of: v → A, G → R, G → S, A → P, A → R, A → D, A → H, A → N, A → G, S → D, P → H, M → D, G → E, I → A, I → V, H → N and/or Q → K.
16. The method of any one of the preceding items, wherein the amino acid substitution is selected from G185R, a186R, a188R, a188D, a188H, a188N, and/or M191D.
17. The method of any one of the preceding items, wherein the increased stability is a decrease in GRAVY value, an increase in pH stability and/or an increase in temperature stability.
18. The method according to any of the preceding items, wherein the GRAVY value is calculated by dividing the sum of the hydropathic values (indices) of all amino acids by the number of amino acid residues in the sequence (calculation according to Kyte and Doolittle).
19. The method of any one of the preceding items, wherein the GRAVY value is calculated by dividing the sum of the hydropathic values (indices) of all amino acids in the sequence by the total number of amino acids in the sequence.
20. The method of any one of the preceding items, wherein the amino acid is replaced with an amino acid selected from R, D, H, G, N or P.
21. The method according to any one of the preceding items, wherein the method comprises substituting at least one amino acid at the following positions:
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 184 to 190 of
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 201 to 208 of
and wherein the amino acid is replaced with an amino acid selected from R, D, H, G, N or P.
22. An α/β -hydrolase obtainable by the method of any one of the preceding items.
23.α/β -hydrolase, which α/β -hydrolase comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, 3,4,5 and/or comprises a sequence corresponding to positions 144 to 217 of SEQ ID NO:2 and/or comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO:6 and/or comprises a sequence having 58% or more sequence identity to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, 3,4,5 and/or comprises a sequence having 58% or more sequence identity to a sequence corresponding to positions 144 to 217 of SEQ ID NO:2 and/or comprises a sequence having 58% or more sequence identity to a sequence corresponding to positions 161 to 235 of SEQ ID NO:6 (CAP-domain; 58% identity to the CAP-domain of SEQ ID NO:1, 2, 3,4,5, 6),
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein said α/β -hydrolase has a GRAVY value that is at least 0.6% less negative than the GRAVY value of a α/β -hydrolase having the polypeptide sequence of SEQ ID NO: 1.
24. An α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO. 6, or a sequence having greater than 58% sequence identity to a sequence corresponding to positions 161 to 235 of SEQ ID NO. 6,
wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein said α/β -hydrolase has a GRAVY value that is at least 0.6% less negative than the GRAVY value of a α/β -hydrolase having the polypeptide sequence of SEQ ID NO: 6.
25. α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, 3,4,5 and/or which comprises a sequence corresponding to positions 144 to 217 of SEQ ID NO:2 and/or which comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO:6 and/or which comprises a sequence having a sequence identity of 58% or more to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, 3,4,5 and/or which comprises a sequence having a sequence identity of 58% or more to a sequence corresponding to positions 144 to 217 of SEQ ID NO:2 and/or which comprises a sequence having a sequence identity of 58% or more to a sequence corresponding to positions 161 to 235 of SEQ ID NO:6 (CAP-domain; 58% identity to the CAP-domain of SEQ ID NO:1, 2, 3,4,5, 6) comprising
Wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 184 to 190 of
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 201 to 208 of
wherein the amino acid substitution is selected from V → A, G → R, G → S, A → P, A → R, A → D, A → H, A → N, A → G, S → D, P → H, M → D, G → E, I → A, I → V, H → N, Q → K, F → Y and/or V → C, and/or
Wherein the amino acid is replaced with an amino acid selected from P, R, D, H, G or N, preferably the amino acid used is selected from R, D, H, G or N, more preferably the amino acid used is selected from R or N.
26. The α/β -hydrolase of any one of the preceding items, wherein the α/β -hydrolase comprises the following amino acid substitutions:
-G → R and a → N, preferably G185R and a 188N;
-G → S and a → R, preferably G185S and a 188R;
-G → R, A → R, A → H, S → D, P → H and M → D, preferably G185R, a186R, a188H, S189D, P190H and M191D;
-V → A, G → R, A → N, G → E, I → A, H → N and Q → K, preferably V160A, G185R, a188N, G199E, I200A, H203N and Q205K;
-V → A, G → S, A → R, G → E, I → A, H → N and Q → K, preferably V160A, G185S, a188R, G199E, I200A, H203N and Q205K;
-V → A, G → E, I → A, H → N and Q → K, preferably V160A, G199E, I200A, H203N and Q205K;
-V → A, G → R, A → R, A → H, G → E, I → A, H → N and Q → K, preferably V160A, G185R, a186R, a188H, G199E, I200A, H203N and Q205K, and/or
-V → A, G → R, A → R, A → H, G → E, I → V, H → N and Q → K, preferably V160A, G185R, a186R, a188H, G199E, I200V, H203N and Q205K;
-V → A, G → R, A → R, A → H, S → D, P → H, M → D, G → E, I → V, H → N and Q → K, preferably V160A, G185R, a186R, a188H, S189D, P190H, M191D, G199E, I200V, H203N and Q205K; and/or
-F → Y and V → C, preferably F183Y and V197C.
27. Use of the α/β -hydrolase of any of the preceding items for degrading Zearalenone (ZEN).
28. A composition comprising the α/β -hydrolase of any one of the preceding items, preferably the composition is a food or feed additive or a food or feed product.
29. The α/β -hydrolase or the composition of any one of the preceding items, for use in the treatment or prevention of a disease.
30. A kit comprising the α/β -hydrolase or the composition of any one of the preceding items.
31. A method for increasing the stability of α/β -hydrolase, said α/β -hydrolase comprising a sequence corresponding to
replacing at least one amino acid at the following positions:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein the amino acid is replaced with an amino acid that: the amino acids used have a more negative hydrophilicity index than the amino acids being replaced,
wherein the hydropathic index is determined by Kyte and Doolittle hydropathic index,
α/β -hydrolase having increased stability is thus obtained.
32. A method for increasing the stability of α/β -hydrolase, said α/β -hydrolase comprising the sequence of
Replacing at least one amino acid at the following positions:
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein the amino acid is replaced with an amino acid that: the amino acids used have a more negative hydrophilicity index than the amino acids being replaced,
wherein the hydropathic index is determined by Kyte and Doolittle hydropathic index,
α/β -hydrolase having increased stability is thus obtained.
33. An α/β -hydrolase, which α/β -hydrolase comprises a sequence corresponding to
-positions corresponding to
Positions corresponding to positions 159 to 204 of
-positions corresponding to
Positions corresponding to positions 176 to 222 of
wherein said α/β -hydrolase has a GRAVY value that is at least 0.6% less negative than the GRAVY value of a α/β -hydrolase having the polypeptide sequence of SEQ ID NO: 1.
34. α/β -hydrolase having a polypeptide sequence which comprises a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, 3,4,5 and/or which comprises a sequence corresponding to positions 144 to 217 of SEQ ID NO:2 and/or which comprises a sequence corresponding to positions 161 to 235 of SEQ ID NO:6 and/or which comprises a sequence having a sequence identity of 58% or more to a sequence corresponding to positions 145 to 218 of SEQ ID NO:1, 3,4,5 and/or which comprises a sequence having a sequence identity of 58% or more to a sequence corresponding to positions 144 to 217 of SEQ ID NO:2 and/or which comprises a sequence having a sequence identity of 58% or more to a sequence corresponding to positions 161 to 235 of SEQ ID NO:6 (CAP-domain; 58% identity to the CAP-domain of SEQ ID NO:1, 2, 3,4,5, 6) comprising
Wherein the polypeptide sequence comprises at least one amino acid substitution at a position:
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 184 to 190 of
-positions corresponding to positions 185 to 191 of
Positions corresponding to positions 201 to 208 of
wherein the amino acid substitution is selected from V → A, G → R, G → S, A → P, A → R, A → D, A → H, A → N, A → G, S → D, P → H, M → D, G → E, I → A, I → V, H → N and Q → K, and/or
Wherein the amino acid is replaced with an amino acid selected from P, R, D, H, G or N, preferably the amino acid used is selected from R, D, H, G or N, more preferably the amino acid used is selected from R or N.
****
It is noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes one or more of such different agents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that may modify or replace the methods described herein.
The term "at least" preceding a series of elements is to be understood as referring to each element in the series, unless otherwise indicated. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The present invention is intended to cover such equivalents.
As used herein, the term "and/or" includes the meaning of "and", "or" and "all or any other combination of elements connected by the term".
The term "less than" or the antisense word "greater than" does not include a particular number.
For example, less than 20 means less than the number shown. Similarly, greater than or higher than means greater than or higher than the number shown, e.g., greater than 80% means greater than or higher than 80% of the number shown.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The term "comprising" as used herein can be substituted with the term "containing" or sometimes with the term "having" as used herein. As used herein, "consisting of" excludes any element, step, or ingredient not specified.
The term "including" means "including but not limited to". The terms "including" and "including but not limited to" are used interchangeably.
It is to be understood that this invention is not limited to the particular methodology, procedures, materials, reagents, materials, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined only by the claims.
All publications (including all patents, patent applications, scientific publications, specifications, etc.) cited throughout the text of this specification are incorporated herein by reference in their entirety, whether supra or infra. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that the incorporated material contradicts or is inconsistent with the present specification, the present specification will take precedence over any such material.
The contents of all documents and patent documents cited herein are incorporated herein in their entirety.
The following sequences are used in this application.
Table 2 sequences used in this application The motifs that can be located in The VI-domain side region described herein are shown in SEQ ID NO:7 and 8 The motifs that can be located in The CAP-loop side region described herein are shown in SEQ ID NO:9, 10, 11 The "X" in The motif can be any amino acid The second amino acid in The motif of SEQ ID NO:9 can be F or Y, denoted "(F/Y)". both The VI-domain and The CAP-loop are contained in The CAP-domain, as described for example by Ollis et al, (1992) 'The alpha/beta hydrosilase fold' Protein Eng.5(3):197-211 and The CAP-domain can be located in The offset (extension) between The b6 and aD of β -fold and α -helices such as α/β -hydrolase.the VI-domain and The CAP-loop are shown underlined in SEQ ID NO: 1-6.
The invention and its advantages are better understood by the following examples, which are given for illustrative purposes only. These examples are not intended to limit the scope of the present invention in any way.
Examples of the invention
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