CN110923222B - Novel alkaline protease acid mutant from bacillus licheniformis - Google Patents
Novel alkaline protease acid mutant from bacillus licheniformisDownload PDFInfo
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Abstract
Translated fromChineseDescription
Technical Field
The invention belongs to the field of protein engineering modification, and particularly relates to a novel alkaline protease mutant protein.
Background
The alkaline protease (alkaline protease) is an enzyme capable of hydrolyzing peptide bonds of proteins under alkaline conditions (the pH is within the range of 9-11), the main component of the alkaline protease is endoprotease, the catalytic site is serine, and the alkaline protease is widely applied to industries such as detergents, foods, medical treatment, brewing, silk and leather making. The alkaline protease adopted at present is a proteolytic enzyme which is bred by a bacterial protoplast mutagenesis method and is derived from bacillus subtilis 2709 through submerged fermentation, extraction and refining, belongs to serine alkaline protease, can hydrolyze a protein molecular peptide chain to generate polypeptide or amino acid, and has strong capability of decomposing protein. The production process adopts the advanced technologies of microfiltration and ultrafiltration membrane separation, spray drying or vacuum freeze drying and the like. The alkaline protease is a popular washing additive in the current market, can greatly improve the washing and dirt removing capacity, particularly has unique washing effect on protein dirt such as blood stains, sweat stains, milk stains, oil stains and the like, is an enzyme occupying the largest proportion in industrial enzymes, and accounts for about 60 percent of the total annual sales volume all over the world.
Site-directed mutagenesis is the introduction of desired changes (usually changes that characterize favorable orientations) including base additions, deletions, point mutations, and the like, by Polymerase Chain Reaction (PCR) or the like, into a DNA fragment (which may be a genome or a plasmid) encoding a protein of interest. The site-directed mutation can rapidly and efficiently improve the character and the characterization of target protein expressed by DNA, and is a very useful means in gene research work. The in vitro site-directed mutagenesis technology is an important experimental means in the research of various fields of biology and medicine at present, is a convenient scheme for modifying and optimizing genes, and is a powerful tool for researching the complex relationship between the structure and the function of protein. The corresponding amino acid sequence and protein structure can be changed by site-directed alteration, deletion or insertion of specific base of a known gene, and the research of the expression product of mutant gene is helpful for human to understand the relationship between protein structure and function and investigate the structure/structural domain of protein. In recent years, the application of site-directed mutagenesis technology of enzyme mainly focuses on the aspects of improving the catalytic activity of enzyme, improving the substrate specificity, improving the thermal stability, enantioselectivity and the like. The site-directed mutagenesis technology of the enzyme opens up a new way for the structure and the function of the enzyme and has great success in the fields of industry, agriculture, food industry, environment and the like.
Disclosure of Invention
The invention provides an alkaline protease mutant with obviously improved enzyme activity under a slightly acidic condition for solving the problems in the prior art. The mutant is obtained by starting from a mature peptide of an alkaline protease gene subC of Bacillus licheniformis (Bacillus licheniformis) and applying a site-directed mutagenesis technology, and is expressed in the Bacillus subtilis.
The invention provides an alkaline protease mutant, wherein the 331 st amino acid of the alkaline protease with the amino acid sequence of SEQ ID NO. 1 is changed from Val to Ile, and the amino acid sequence of the mutant is SEQ ID NO. 2.
The invention provides an alkaline protease mutant, wherein the 339 th amino acid of the alkaline protease with the amino acid sequence of SEQ ID NO. 2 is changed into His from Leu, and the amino acid sequence of the mutant is SEQ ID NO. 3.
The invention is based on the alkaline protease subC from the bacillus licheniformis, obtains two alkaline protease mutants subC M pH1 and subC M pH2 by the site-directed mutagenesis technology, and ferments the mutants in the bacillus subtilis, the enzyme activity of the fermentation supernatant is 3675U/mL and 3767U/mL respectively under the condition ofpH 6, and the enzyme activity level is obviously improved compared with the enzyme activity level (1198U/mL) of the bacillus subtilis expressing wild type alkaline protease subC under the same condition.
Drawings
FIG. 1: a plasmid map of the alkaline protease expression vector;
FIG. 2: the bar chart of relative enzyme activity of the alkaline protease mutant after heat treatment.
Detailed Description
The methods of the invention are further illustrated below by way of examples, in which experimental procedures not specifying the conditions are generally run under conventional conditions, e.g., as described in molecular cloning, a laboratory Manual, written by J. Sambruke (Sambrook), et al, or as recommended by the manufacturer. The present invention may be better understood and appreciated by those skilled in the art with reference to the following examples. However, the method of carrying out the present invention should not be limited to the specific method steps described in the examples of the present invention.
The terms and associated assay methods referred to in the present invention are explained below:
1. the protease activity determination method comprises the following steps: adopts the method for determining the protease preparation of the national standard of the people's republic of China (GB/T25327-2009).
2. Definition of enzyme activity unit: 1g of solid enzyme powder (or 1mL of liquid enzyme) hydrolyzes casein for 1min under the conditions of certain temperature and pH value to generate 1 mu g of tyrosine, namely 1 enzyme activity unit expressed by U/g (U/mL).
3. Alkaline protease the activity of the protease was determined using the forskolin method using a solution comprising: folin use solution (one commercial Folin solution was mixed with two portions of water, shaken up), sodium carbonate solution (42.4g/L), trichloroacetic acid (65.4g/L), gradient pH buffer, casein solution (10.0 g/L). The reaction process is as follows: adding 1mL enzyme solution into the test tube, performing warm bath at 40 deg.C for 2min, adding 1mL casein solution, shaking, performing warm bath at 40 deg.C for 10min, adding 2mL trichloroacetic acid solution, and shaking (adding trichloroacetic acid and casein solution into blank control). Taking out and standing for 10min, and filtering with slow qualitative filter paper. Taking 1mL of filtrate, adding 5mL of sodium carbonate solution, adding 1mL of forskolin reagent solution, developing at 40 ℃ for 20min, and measuring absorbance at 680nm wavelength by using a 10mm cuvette.
4. The identification of the alkaline protease mutant refers to the amino acid mutated in the alkaline protease mutant by the "amino acid substituted at the original amino acid position". Such as Val331Ile, indicating the substitution of the amino acid in position 331 from Val of the parent alkaline protease to Ile, the numbering of the positions corresponding to the numbering in SEQ ID NO 1 of the appendix sequence Listing. For example, Val331Ile/Leu339His indicates that the amino acids at positions 331 and 339 are mutated.
Example 1 construction of alkaline protease subcoC expression vector and recombinant Strain
The vector backbone sequence to be cloned was obtained by amplifying the vector DNA sequence by PCR using the pMA5 plasmid as a template. Taking Bacillus licheniformis genome DNA as a template, amplifying a subcoC complete gene by PCR to obtain an alkaline protease sequence carrying a signal peptide and a mature peptide, wherein the coded amino acid sequence is SEQ ID NO: 1. the PCR amplification conditions are as follows: 10min at 94 ℃; 60s at 94 ℃, 60s at 58 ℃, 2min at 72 ℃ and 30 cycles; 10min at 72 ℃. The PCR amplification product was recovered using the E.Z.N.A.gel Extraction Kit. Overlapping extension PCR is carried out on the recovered enzyme gene sequence and the vector skeleton sequence to form a polymer, and an amplification system is as follows: 5 XPPhusion HF Buffer 10. mu.L, 2.5mM dNTPs 8. mu.L, gene fragment (subcoC fragment) 4. mu.L, vector backbone fragment (pMA5) 6. mu.L, Phusion DNA Polymerase 1. mu.L, ddH2O21. mu.L. The amplification condition is 98 ℃ for 10 min; 10s at 98 ℃, 3min at 72 ℃ and 20 cycles; 10s at 98 ℃, 6min at 72 ℃ and 15 cycles; 10min at 72 ℃. And transforming the polymer into Bacillus subtilis 1A751 host bacteria, screening positive transformants to obtain a recombinant strain expressing wild type alkaline protease subcoC, and naming the recombinant strain as Bacillus subtilis subcoC. A plasmid in the Bacillus subtilis subcoC is extracted and named as pMA 5-subcoC (containing a wild-type subcoC gene), and the plasmid map of the plasmid is shown in figure 1.
The Bacillus subtilis 1A751 is transformed by a competent method, and the specific transformation process is as follows: freshly activated B.subtilis 1A751 was plated on LB (tryptone 1%, yeast powder 0.5%, NaCl 1%) plates into 5ml of GM I (GM I formulated in 1X minimum salt solution 95.6ml, 20% dextrose2.5ml of glucose, 0.4ml of 5% hydrolyzed casein and 1ml of 10% yeast powder juice; wherein, the preparation method of the lowest salt solution comprises the following steps: k2HPO4 14g/L，KH2PO46g/L，(NH4)2SO42g/L, trisodium citrate 1g/L, MgSO4·7H2O0.2 g/L, the solution was dissolved in distilled water successively, and cultured overnight at 30 ℃ with shaking at 125 rpm. The next day, 2ml of the culture broth was transferred to 18ml of GM I and cultured at 37 ℃ and 250rpm for 3.5 hours. Then 10ml of the culture fluid from the previous step was transferred to 90ml of GM II (GMII preparation method: 96.98ml of 1 × minimum salt solution, 2.5ml of 20% glucose, 0.08ml of 5% hydrolyzed casein, 0.04ml of 10% yeast powder juice, 1M MgCl2 0.25ml，1M CaCl20.05ml of the culture medium was cultured at 37 ℃ and 125rpm for 90 minutes, and then centrifuged at 5000g for 10 minutes to collect the cells. And (3) lightly suspending the thalli by using 10ml of original culture solution supernatant, wherein the suspended thalli are competent cells. Then, an appropriate amount of DNA was added to 0.5ml of the competence, and the mixture was subjected to shaking culture at 37 ℃ and 200rpm for 30min, followed by plating, further overnight culture at 37 ℃ and examination and verification of transformants the next day.
Example 2 construction of alkaline protease mutants Using Point mutation technique
The nucleotide mutation is introduced into the basic protease subcoC gene from the bacillus licheniformis by utilizing a point mutation technology, and the amplification system of the Val331Ile site mutation PCR is as follows: 5 XPPhusion HF Buffer 10 uL, 2.5mM dNTPs 8 uL, template DNA pMA 5-subcoC plasmid (100 ng/. mu.L) 1 uL, Phusion DNA Polymerase 1 uL, ddH2O28. mu.L, upstream mutation primer 5'-gccttggcgcaaaccgtagtttacggcgaacctctcattaaagcggacaa-3', downstream mutation primer 5'-ttgtccgctttaatgagaggttcgccgtaagtgagggtttgcgccaaggc-3'. The reaction conditions are as follows: 10min at 98 ℃; 10s at 98 ℃, 20s at 55 ℃, 3min at 72 ℃ and 20 cycles; 10min at 72 ℃. After the PCR product is transformed into escherichia coli DH5 alpha competent cells to obtain positive transformants, plasmids are extracted by an E.Z.N.A.plasmid Extraction Kit and named pMA 5-subBCMpH 1. The amplification system of Val331Ile/Leu339His overlapping site mutation PCR is as follows: 5 XPPhusion HF Buffer 10 uL, 2.5mM dNTPs 8 uL, template DNA pMA 5-subcocMpH 1 plasmid (100 ng/. mu.L) 1 uL, Phusion DNA Polymerase 1 uL, ddH2O28. mu.L, upstream mutation primer 5' -gccgtcctgcatcaggagagctaacaagcttctcatccggactt-3 ', downstream mutation primer 5'-aagtccggatgagaagcatgctagccctgtatccaggacggc-3'. The reaction conditions are as follows: 10min at 98 ℃; 10s at 98 ℃, 20s at 55 ℃, 3min at 72 ℃ and 20 cycles; 10min at 72 ℃. After the PCR product is transformed into escherichia coli DH5 alpha competent cells to obtain positive transformants, plasmids are extracted by an E.Z.N.A.plasmid Extraction Kit and named pMA 5-subBCMpH 2. The plasmids pMA 5-subbCMpH 1 and pMA 5-subbCMpH 2 are transformed into Bacillus subtilis 1A751 host bacteria, positive transformants are screened to obtain recombinant strains expressing wild type alkaline protease subbC mutants, and the recombinant strains are respectively named as Bacillus subtilis subbCMpH 1(Bacillus subtilis subMpH 1) and subbCMpH 2(Bacillus subtilis subbCMpH 2).
EXAMPLE 3 evaluation of alkaline protease mutants
3.1 Shake flask fermentation
The 2 alkaline protease mutant recombinant strains (subBCMpH 1 and subBCMpH 2) and the control bacterium Bacillus subtilis subbC constructed above were inoculated into 50mL seed media (yeast extract 0.5%, tryptone 0.5%, glucose 1%, K)2HPO41.8%, kanamycin 25. mu.g/mL), at 34 ℃ for 8h with shaking at 210 rpm. Then inoculating 2.5mL of fermentation liquor into 50mL of fermentation medium (1-2% of yeast powder, 2-5% of bean cake powder, 5-10% of maltodextrin, 0.1-0.5% of sodium citrate, CaCl)2 0.1～0.5％，MgSO4 0.1～0.5％，K2HPO40.5-2%), at 34 ℃ and 250rpm for 72 h; centrifuging and taking supernatant.
3.2 enzyme Activity assay
The alkaline protease enzyme activity of the 2 recombinant strains (subCM1 and subCM2) and the control bacillus subtilis subbC fermentation supernatant under the condition ofpH 6 is respectively detected by adopting a national standard protease preparation determination method (GB/T25327-2009) of the people's republic of China at the temperature of 37 ℃. The enzyme activity of the fermentation supernatant is 3675U/mL and 3767U/mL respectively under the condition ofpH 6, the enzyme activity is obviously improved compared with the enzyme activity (1198U/mL) of the bacillus subtilis expressing wild alkaline protease subcoC under the same condition, and the enzyme activity data are shown in figure 2.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> a novel alkaline protease acid mutant derived from Bacillus licheniformis
<130> 2019
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 379
<212> PRT
<213> Bacillus licheniformis
<400> 1
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Thr Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Ser Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 2
<211> 379
<212> PRT
<213> Bacillus licheniformis
<400> 2
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Thr Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Ser Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Ile Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 3
<211> 379
<212> PRT
<213> Bacillus licheniformis
<400> 3
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Thr Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Ser Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Ile Ala Gly Ala Ala Ala
325 330 335
Leu Ile His Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
Claims (5)
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| Enhanced Production of Alkaline Protease by a Mutant of Bacillus licheniformis N-2 for Dehairing;Muhammad Nadeem;《BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY》;20101031;第53卷(第5期);第1015-1025页* |
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