Leader peptide mutant and application thereof in keratinase production
阅读说明：本技术 一种前导肽突变体及其在角蛋白酶生产中的应用 (Leader peptide mutant and application thereof in keratinase production ) 是由 张娟 彭政 陈坚 堵国成 于 2019-11-22 设计创作，主要内容包括：本发明公开了一种前导肽突变体及其在角蛋白酶生产中的应用,属于酶工程和微生物工程技术领域。本发明通过将角蛋白酶的前导肽进行突变大大提高了角蛋白酶的产量；将本发明的以由编码信号肽的基因、编码前导肽突变体的基因以及编码角蛋白酶的基因依次串联而得的DNA片段为目的基因,以pP43NMK质粒为表达载体,以枯草芽孢杆菌(Bacillus subtilis)WB600为表达宿主构建得到的可高产角蛋白酶的枯草芽孢杆菌工程菌进行发酵,发酵24h,即可使得发酵上清液中的酶活高达109290～157080U/mL。(The invention discloses a leader peptide mutant and application thereof in keratinase production, belonging to the technical field of enzyme engineering and microbial engineering. The invention greatly improves the yield of the keratinase by mutating the leader peptide of the keratinase; the Bacillus subtilis engineering bacteria capable of producing keratinase with high yield, which is constructed by taking a gene coding a signal peptide, a gene coding a leader peptide mutant and a gene coding keratinase which are connected in series in sequence as a target gene, taking a pP43NMK plasmid as an expression vector and taking Bacillus subtilis WB600 as an expression host, is fermented for 24 hours, so that the enzyme activity in the fermented supernatant can reach 109290-157080U/mL.)
1. A leader peptide mutant which is obtained by mutating leucine 76, aspartic acid 39, serine 17, glycine 18, aspartic acid 37, alanine 48 or glutamic acid 57 of a leader peptide having an original amino acid sequence shown in SEQ ID NO. 1.
2. The leader peptide mutant according to claim 1, wherein the leader peptide mutant is obtained by mutating leucine at position 76 to alanine, leucine at position 76 to valine, leucine at position 76 to methionine, leucine at position 76 to phenylalanine, aspartic acid at position 39 to serine, serine at position 17 to alanine, glycine at position 18 to serine, aspartic acid at position 37 to glutamic acid, alanine at position 48 to valine, or glutamic acid at position 57 to lysine of the leader peptide having an original amino acid sequence shown in SEQ ID No. 1.
3. A gene encoding the leader peptide mutant according to claim 1 or 2.
4. A nucleotide sequence obtained by connecting in series the nucleotide sequence of a gene encoding a signal peptide, the nucleotide sequence of a gene encoding a leader peptide mutant according to claim 1 or 2, and the nucleotide sequence of a gene encoding keratinase in this order.
5. A nucleotide sequence as claimed in claim 4, in which the nucleotide sequence of the gene encoding keratinase is as shown in SEQ ID No. 3.
6. A recombinant plasmid carrying the nucleotide sequence of claim 4 or 5.
7. A host cell carrying the nucleotide sequence of claim 4 or 5 or the recombinant plasmid of claim 6.
8. A method for producing keratinase, which comprises inoculating the host cell of claim 7 into a fermentation medium to perform fermentation to obtain a fermentation broth; centrifuging the fermentation liquor to obtain fermentation supernatant; extracting the fermentation supernatant to obtain keratinase.
9. The method of claim 8, wherein the fermentation medium comprises peptone 20g/L, yeast powder 10g/L, sucrose 20g/L, KH2PO43 g/L、Na2HPO46g/L and MgSO40.3g/L。
10. Use of a leader peptide mutant according to claim 1 or 2, a gene according to claim 3, a nucleotide sequence according to claim 4 or 5, a recombinant plasmid according to claim 6, a host cell according to claim 7 or a method according to claim 8 or 9 for the production of keratinase.
Technical Field
The invention relates to a leader peptide mutant and application thereof in keratinase production, belonging to the technical field of enzyme engineering and microbial engineering.
Background
Keratinase is a specific protease capable of degrading insoluble sclerostin, keratin-like substrates (e.g., feather, wool, hair, dander, etc.), which is mainly obtained by growth and secretion of microorganisms such as bacteria, fungi, actinomycetes, etc., while using keratin as a single carbon nitrogen source.
As a protease with wider substrate specificity and strong hydrolysis catalytic capability, the keratinase can replace the traditional protease, is widely applied to the fields of feather degradation, leather textile, feed additives, organic fertilizers, detergents and the like, and has huge market.
However, wild keratinase has poor performance and low yield, and is far from meeting the market demand, so that it is difficult to be really applied to industrial production.
Currently, means for constructing genetically engineered bacteria are often adopted to enhance the transcription and translation of keratinase genes so as to achieve the purpose of improving the yield of keratinase, for example, Porres J M et al constructed a genetically engineered bacterium capable of producing keratinase by using a gene encoding keratinase derived from B.licheniformis MKU3 as a target gene, pPICZ α A as a vector and Pichia pastoris X33 as a host, and fermented for 24h so as to achieve only 285U/L of the enzyme activity in the fermentation supernatant (see specifically: Porres J M, Benito M J, Lei X G. functional expression of keratinase (fire) gene in Pileaplast. Biotechnology Letters [ J, 2002,24(8): 636, 636), 636, etc. to achieve the production of proteins encoded by Bacillus licheniformis in Pileaplastograph. Biotechnology Letters, b, see that the genetically engineered gene encoding proteins produced by Bacillus subtilis, Zynchus, Zostriatron b, and Zostrich J.7. 7. fermentation, 11. the production of protein produced by using Bacillus subtilis, Zymobacter strain, Zymomonas a strain, and Zymobacter 11. fermentation medium, and fermentation.
Therefore, it is urgently needed to find a method for improving the yield of keratinase to meet the requirement of industrial production.
Disclosure of Invention
[ problem ] to
The technical problem to be solved by the invention is to provide a method for producing keratinase with high yield.
[ solution ]
In order to solve the technical problems, the invention provides a leader peptide mutant, which is obtained by mutating leucine 76, aspartic acid 39,
In one embodiment of the present invention, the leader peptide mutant is obtained by mutating leucine at position 76 to alanine, leucine at position 76 to valine, leucine at position 76 to methionine, leucine at position 76 to phenylalanine, aspartic acid at position 39 to serine, serine at
In one embodiment of the invention, the nucleotide sequence encoding the leader peptide is shown in SEQ ID No. 2.
The invention also provides a gene for coding the leader peptide mutant.
The invention also provides a nucleotide sequence which is obtained by sequentially connecting the nucleotide sequence of the gene for coding the signal peptide, the nucleotide sequence of the gene for coding the leader peptide mutant and the nucleotide sequence of the gene for coding the keratinase in series.
In one embodiment of the invention, the nucleotide sequence of the gene encoding keratinase is shown in SEQ ID No. 3.
In one embodiment of the invention, the amino acid sequence of the keratinase is shown in SEQ ID No. 4.
In one embodiment of the present invention, the nucleotide sequence of the gene encoding the signal peptide is shown in SEQ ID No. 5.
In one embodiment of the invention, the amino acid sequence of the signal peptide is shown in SEQ ID No. 6.
The invention also provides a recombinant plasmid carrying the nucleotide sequence.
In one embodiment of the present invention, the vector of the recombinant plasmid is a pP43NMK plasmid.
The invention also provides a host cell carrying the nucleotide sequence or the recombinant plasmid.
In one embodiment of the invention, the host cell is a bacterium or a fungus.
In one embodiment of the invention, the host cell is Bacillus subtilis.
In one embodiment of the invention, the host cell is Bacillus subtilis WB 600.
The invention also provides a method for producing keratinase, which comprises the steps of inoculating the host cell into a fermentation medium for fermentation to obtain a fermentation liquid; centrifuging the fermentation liquor to obtain fermentation supernatant; extracting the fermentation supernatant to obtain keratinase.
In one embodiment of the invention, the components of the fermentation medium comprise peptone 20g/L, yeast powder 10g/L, sucrose 20g/L, KH2PO43g/L、Na2HPO46g/L and MgSO40.3g/L。
In one embodiment of the invention, the temperature of the fermentation is 37 ℃ and the rotation speed is 220 rpm.
The invention also provides the application of the leader peptide mutant, the gene, the nucleotide sequence, the recombinant plasmid, the host cell or the method in the production of keratinase.
[ advantageous effects ]
The invention greatly improves the yield of the keratinase by mutating the leader peptide of the keratinase; the bacillus subtilis engineering bacteria capable of producing keratinase with high yield, which is constructed by taking a gene coding a signal peptide, a gene coding a leader peptide mutant and a gene coding keratinase which are connected in series in sequence as a target gene, taking a pP43NMK plasmid as an expression vector and taking bacillus subtilis WB600 as an expression host, is fermented for 24 hours, so that the enzyme activity in the fermented supernatant can reach 109290-157080U/mL.
Drawings
FIG. 1: the effect of an amino acid substitution at leader cleavage site P1 (i.e., amino acid 76) on the catalytic activity of keratinase;
FIG. 2: influence of site-directed mutagenesis of leader peptide on the catalytic activity of keratinase.
Detailed Description
Coli JM109 referred to in the examples below was purchased from North Naphthora; the pP43NMK plasmids referred to in the examples below were purchased from a haloghic organism; bacillus subtilis WB600 mentioned in the examples below is described in patent application publication No. CN 102492645A.
The media referred to in the following examples are:
LB liquid medium: yeast powder 5.0 g.L-1Tryptone 10.0 g.L-1、NaCl 10.0g·L-1Kanamycin 100 mg. L-1。
LB solid medium: yeast powder 5.0 g.L-1Tryptone 10.0 g.L-1、NaCl 10.0g·L-115g/L agar powder and 50 mg/L kanamycin-1。
Seed culture medium: yeast powder 5 g.L-1Peptone 10 g. L-1、NaCl 5g·L-1。
Fermentation medium:
The detection methods referred to in the following examples are as follows:
measurement of the enzyme activity of keratinase: taking 50 μ L of the fermentation supernatant diluted properly, adding 150 μ L of 50mM Gly/NaOH solution as buffer and 100 μ L of 2.5% water-soluble keratin (purchased from Taishiai (Shanghai) chemical industry development Co., Ltd., product code: K0043) as substrate, mixing, and reacting at 40 deg.C for 20 min; the reaction was stopped by adding 200. mu.L of 4% (w/v) trichloroacetic acid (TCA) and centrifuged at 8000r/min at room temperature for 3 min. The supernatant was taken to 200. mu.L, and 1mL of 4% (w/v) Na was added2CO3Mixing with 200 μ L of Folin phenol reagent, mixing, developing at 50 deg.C for 10min, and measuring clear solution light absorption value at 660nm with 0.5cm quartz cuvette; 3 experimental groups are paralleled, the blank control is that the reaction terminator TCA is added before the substrate is added, and the rest operations are the same as above;
definition of enzyme activity: OD under this condition660The enzyme amount required is 0.001 per liter and is one enzyme activity unit (1U).
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