CN112662654A - Alkaline protease mutant and application thereof - Google Patents
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Abstract
The invention mainly uses DNaseI to digest alkaline protease genes from Bacillus licheniformis and Bacillus clausii through a DNA shuffling technology, purifies and recovers target fragments, constructs an alkaline protease mutation library through primer-free PCR and primer-containing PCR, and screens to obtain a mutant with remarkably improved alkaline protease activity, wherein the amino acid sequence of the mutant is shown as SEQ ID NO: and 6. Under the appropriate conditions, the activity of the mutant alkaline protease is 61.75U/mL, which is obviously improved compared with the wild type.
Description
Technical Field
The invention belongs to the technical field of microorganisms and genetic engineering, and particularly relates to an alkaline protease mutant and application thereof.
Background
Protease is a hydrolase with a complex structure and function, and is capable of cleaving peptide bonds to produce short peptides or amino acids. Proteases can be classified into three types according to their optimum pH: the optimal pH value of the acidic protease is 2.0-5.0, and the acidic protease is mainly derived from fungi; neutral protease with optimum pH of 7.0, mainly derived from plant; alkaline protease, having an optimum pH of 8.0 or more, is mainly derived from microorganisms. Protease is one of the most important industrial enzyme preparations, and has been widely used in the fields of detergents, medicines, foods, leather, silk, photography, etc., because the sale amount of protease accounts for 60% or more of the total sale amount of all enzyme preparations.
Synthetic detergents have been used as cleaning agents for over 100 years. Currently, synthetic detergents have become an integral part of people's daily lives around the world. However, the negative impact of ingredients such as phosphate in synthetic detergents on the ecosystem has become increasingly appreciated. Therefore, detergent enzymes have rapidly developed into a wide range of fields including proteases, lipases, amylases and cellulases as environmentally friendly substitutes for harmful chemicals in detergents. Enzymes commonly used in the detergent industry are alkaline enzymes, since the pH of detergents is typically between 9.0 and 12.0. Among alkaline enzymes used in detergents, alkaline protease accounts for the first place, and accounts for 40% of the global protease sales. Therefore, the development of a high-activity alkaline protease contributes to the reduction of the cost of detergent protease and the improvement of the production efficiency of detergent protease.
Directed evolution, also called irrational design, does not need to rely on reliable protein structure and structure-function relationship, but simulates natural evolution mechanism in laboratory, and then constructs enzyme molecule in vitro evolution conditions, and directed screening to obtain mutants with ideal performance. The DNA shuffling technology is one of directed evolution technologies, and can break genes into segments with certain sizes by using DNaseI, and mismatch occurs among the segments by primer-free PCR (polymerase chain reaction), so that a large mutation library is constructed.
Disclosure of Invention
In view of the disadvantages of the prior art, the present invention aims to provide an alkaline protease mutant with improved alkaline protease activity. The invention mainly uses DNaseI to digest alkaline protease genes from Bacillus licheniformis and Bacillus clausii through a DNA shuffling technology, purifies and recovers target fragments, constructs an alkaline protease mutation library through primer-free PCR and primer-containing PCR, and screens to obtain mutants with obviously improved alkaline protease activity.
One of the purposes of the invention is to provide an alkaline protease mutant, wherein the amino acid sequence of the alkaline protease mutant is shown as SEQ ID NO: and 6.
Another object of the present invention is to provide a gene encoding the alkaline protease mutant.
In a specific embodiment of the invention, the nucleotide sequence of the gene is as shown in SEQ ID NO: 5, respectively.
It is a further object of the present invention to provide a vector comprising a gene of the alkaline protease mutant. The vector may be one of vectors for producing a protein by gene recombination, such as an expression vector, known to those skilled in the art.
In a specific embodiment of the invention, the vector is pWB 980.
In another aspect, the present invention provides a host cell comprising said encoding gene or vector. The host cell may be any host suitable for producing the alkaline protease mutant of the present invention from the gene or vector of the present invention, for example, Bacillus subtilis.
In a specific embodiment of the invention, the host cell is Bacillus subtilis WB 600.
The fifth purpose of the invention is to provide the use of the alkaline protease mutant of the invention for hydrolyzing peptide bonds of proteins to generate polypeptides or amino acids; and, the alkaline protease mutant has an increased alkaline protease activity as compared to the wild type.
The sixth object of the present invention is to provide a method for preparing the alkaline protease mutant of the present invention by gene recombination and expression using the gene encoding the alkaline protease mutant of the present invention or the vector of the present invention. Genetic recombination methods and expression hosts known to those skilled in the art can be used, and media and culture conditions suitable for expression by the host are selected. The method may further comprise a step of recovering the alkaline protease mutant, which may involve a step of isolating or purifying the alkaline protease mutant from the culture or expression product of the host, and may be carried out using any method known to those skilled in the art.
Has the advantages that:
1. the invention utilizes DNA shuffling technology to carry out directed evolution on alkaline protease from bacillus clausii and alkaline protease from bacillus licheniformis in vitro to obtain alkaline protease mutant PROM with improved enzyme activity, and under appropriate conditions, the activity of the mutant alkaline protease is 61.75U/mL, which is obviously improved compared with parent bacillus clausii CPR (15.63U/mL) and bacillus licheniformis LPR (46.87U/mL).
2. The invention utilizes the gene of the alkaline protease mutant to express in a bacillus expression system to obtain an alkaline protease mutant recombinant strain, utilizes the recombinant strain to ferment and culture, and separates or purifies the fermentation product to obtain the alkaline protease mutant.
Drawings
FIG. 1: comparing the enzyme activities of CPR, LPR and mutant PROM.
FIG. 2: temperature optimum for mutant PROM.
FIG. 3: pH optimum of mutant PROM.
FIG. 4: temperature stability of mutant PROM.
FIG. 5: pH stability of mutant PROM.
FIG. 6: protein glue pattern of mutant PROM and Bacillus licheniformis LPR.
Detailed Description
The invention is further described below by means of specific embodiments. Technical means, materials and the like to which the following embodiments refer may be known to those skilled in the art, and appropriate ones may be selected among known means and materials capable of solving the respective technical problems, unless otherwise specified. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
The following definitions are used in the present invention:
1. nomenclature for amino acid and DNA nucleic acid sequences
The accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
2. Identification of alkaline protease mutants
In the present invention, cpr represents the gene sequence of B.clausii-derived alkaline protease (shown by SEQ ID NO: 1), lpr represents the gene sequence of B.licheniformis-derived alkaline protease (shown by SEQ ID NO: 3), and prom represents the gene sequence of alkaline protease mutant (shown by SEQ ID NO: 5); CPR represents alkaline protease derived from Bacillus clausii (amino acid sequence shown in SEQ ID NO: 2), LPR represents alkaline protease derived from Bacillus licheniformis (amino acid sequence shown in SEQ ID NO: 4), PROM represents alkaline protease mutant (amino acid sequence shown in SEQ ID NO: 6).
The culture medium and the enzyme activity determination method used by the invention are as follows:
LB culture medium: 5g/L of yeast powder, 10g/L of peptone and 5g/L of sodium chloride;
b, bacillus subtilis competence preparation culture medium:
SP-A salt solution: (NH4)2SO4 4g/L，K2HPO4·3H2O 28g/L，KH2PO412g/L, 2g/L sodium citrate;
SP-B salt solution: MgSO (MgSO)4·7H2O 0.4g/L；
100 × CAYE solution: casein hydrolysate 20g/L, yeast powder 100 g/L;
SPI (200 mL): 98mL of SP-A salt solution, 98mL of SP-B salt solution, 2mL of 50% glucose and 2mL of 100 xCAYE;
SPII medium (600 mL): SPI 588mL, 50mmol/L CaCl2 6mL，250mmol/L MgCl26mL；
100 × EGTA solution: 10mmol/L EGTA solution.
Determination of the activity of alkaline protease:
Suc-Ala-Ala-Pro-Phe-pNA (AAPF) was used as substrate and p-nitroaniline (pNA) was used as standard.
Preparation of samples: putting 100 mu L of enzyme solution diluted by distilled water into a 96-well plate, adding 20 mu L of 6mmol/L AAPF solution, mixing uniformly, heating accurately in a water bath at 40 ℃ for 10min, immediately placing on ice to terminate the reaction, and measuring the pNA content in the solution. Each sample was replicated 3 times and the results averaged.
Preparation of a reference substance: the same method as the sample preparation method was used except that the enzyme solution after inactivation was added to the 96-well plate.
Wherein, before the enzyme solution and the AAPF solution are mixed, the two solutions are preheated in a water bath at 40 ℃ for more than 2 min.
Definition of enzyme activity: hydrolysis of AAPF at 40 deg.C per mL enzyme solution produced an enzyme amount of 1. mu. mol pNA per min (U/mL).
The technical scheme of the invention is summarized as follows:
1. respectively digesting an alkaline protease gene from Bacillus clausii (Bacillus clausii) and an alkaline protease gene from Bacillus licheniformis (Bacillus licheniformis) by using DNaseI, and purifying and recovering a target fragment of about 100 bp;
2. constructing a bacillus subtilis recombinant strain containing an alkaline protease mutant gene:
(1) the purified fragments are mutually primer amplified through primer-free PCR;
(2) amplifying alkaline protease mutant genes by PCR with primers;
(3) carrying out enzyme digestion on the coding gene of the alkaline protease mutant, and connecting the coding gene to an expression vector to obtain a recombinant vector carrying the coding gene of the alkaline protease mutant;
(2) transferring the recombinant vector into a bacillus subtilis host WB600, obtaining a recombinant strain through resistance screening, fermenting the recombinant strain through a 96-hole deep-hole plate, and measuring enzyme activity to obtain an alkaline protease mutant with improved alkaline protease activity;
3. preparing an alkaline protease mutant by the recombinant strain through shake flask fermentation;
4. and (3) obtaining the mutant PROM with improved alkaline protease activity by measuring the alkaline protease activity.
5. Sequencing verification shows that the amino acid sequence of the alkaline protease mutant PROM is shown as SEQ ID NO: 6, and the nucleotide sequence of the coding gene prom is shown as SEQ ID NO: 5, respectively.
Example 1: construction of wild-type Alkallikrein pro recombinant Strain
1.1 Synthesis and amplification of wild-type alkaline protease genes cpr and lpr
According to GenBank: FJ940727.1 obtains the sequence of the alkaline protease gene cpr (shown as SEQ ID NO: 1) from the Bacillus clausii, and the sequence is determined according to GenBank: AY590140.1 obtains the sequence of Bacillus licheniformis-derived alkaline protease gene lpr (shown in SEQ ID NO: 3), and entrusts the organism company to synthesize the gene sequence and amplify the gene sequence by PCR, wherein the primer sequences are as follows:
cpr primer P1: F5'-CCCAAGCTTATGAGGAGGGAACCGAATGAAG-3';
cpr primer P2: R5'-CGCGGATCCTTATTGATTAGCGTGTTGCCGC-3';
lpr primer P1: F5'-CCCAAGCTTATGATGAGGAAAAAGAGTTTTTGGC-3';
lpr primer P2: R5'-CGCGGATCCTTATTGAGCGGCAGCTTCGACATTG-3';
carrying out amplification by taking P1 and P2 as upstream and downstream primers;
the reaction system for amplification is as follows:
| upstream primer P1 | 1.5μL |
| Downstream primer P2 | 1.5μL |
| DNA template | 2.0μL |
| Primerstar enzyme | 25μL |
| ddH2O | 20μL |
The setting of the amplification program is as follows: pre-denaturation: 5min at 95 ℃; denaturation: 30s at 95 ℃; annealing: 15s at 56 ℃; extension: 2min at 72 ℃; reacting for 30 cycles; extension: 10min at 72 ℃.
1.2 linearization of expression vectors
The plasmid pWB980 was extracted, and the extraction process was carried out according to the manual of the kit. The product is recovered by a DNA gel recovery kit after agarose gel electrophoresis after HindIII and BamHI double enzyme digestion, and a linearized vector sequence is obtained.
1.3, target fragments (cpr, lpr) subjected to double enzyme digestion by HindIII and BamHI are connected with vector fragments to form recombinant plasmids pWB980-cpr and pWB980-lpr, the recombinant plasmids are transformed into Bacillus subtilis WB600, and the sequences are shown as SEQ ID NO: 1 and SEQ ID NO: 3, respectively.
1.4 Shake flask fermentation
The recombinant strain is inoculated in 5mL LB liquid culture medium (containing kanamycin, 50 ug/mL), cultured overnight at 37 ℃ at 220r/min, transferred to 50mL fresh fermentation LB culture medium according to the inoculum size of 2 percent, cultured for 48h at 37 ℃ at 220r/min, and the supernatant is taken as crude enzyme solution.
Example 2: alkaline protease mutant obtained by DNA shuffling method
2.1 digesting alkaline protease genes cpr and lpr respectively by DNaseI, carrying out agarose gel electrophoresis, and recovering a fragment of about 100bp by a DNA gel recovery kit;
the DNaseI digestion system is as follows:
| DNA | 3μL |
| DNaseI(1U/μL) | 1μL |
| ddH2O | 21μL |
reaction conditions are as follows: digestion was carried out at 15 ℃ for 20min and then at 90 ℃ for 10min to terminate the reaction.
2.2 primer-free PCR
The cpr and lpr purified fragments were amplified as primers for each other, and the obtained PCR product was recovered using a DNA purification recovery kit.
The reaction system for amplification is as follows:
| ipr gene fragment | 10μL |
| cpr gene fragment | 10μL |
| dNTPs | 1μL |
| TaqDNA polymerase | 25μL |
| ddH2O | 4μL |
The amplification conditions were: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30s, annealing at 45 ℃ for 30s, and extension at 72 ℃ for 1min for 55 cycles; extension at 72 ℃ for 10 min.
2.3 construction of alkaline protease mutant libraries
The above-mentioned recovered product was used as a template to construct an alkaline protease mutation library by PCR amplification.
Primers were designed as follows:
primer P1: F5'-CCCAAGCTTATGATGAGGAAAAAGAGTTTTTGGC-3';
primer P2: R5'-CGCGGATCCTTATTGATTAGCGTGTTGCCGC-3';
the reaction system for amplification is as follows:
| DNA template | 1μL |
| Primer P1 | 1μL |
| Primer P2 | 1μL |
| TaqDNA polymerase | 25μL |
| ddH2O | 22μL |
The amplification conditions were: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min for 20 cycles; extension at 72 ℃ for 10 min.
2.4 cloning the obtained alkaline protease mutant genes into an expression vector pWB980 respectively to obtain a plurality of recombinant plasmids pWB980-promx, and transforming the recombinant plasmids pWB980-promx into the bacillus subtilis WB600 to obtain the recombinant strains capable of expressing the alkaline protease mutants.
Example 3: screening of alkaline protease mutants
3.196 hole deep hole plate primary screen
The recombinant strain obtained in example 2 was inoculated on a kanamycin-resistant plate, cultured at 37 ℃ for 12 hours, a single colony was selected and inoculated on 5mL of LB medium (kanamycin-resistant), cultured with shaking at 37 ℃ and 220r/min for 12 hours, then inoculated on a 96-well deep-well plate at an inoculum size of 2%, and cultured with shaking at 37 ℃ and 600r/min for 48 hours, to prepare an alkaline protease solution.
Respectively measuring the alkaline protease activity of the enzyme solution, comparing the enzyme activity of all mutants with that of wild alkaline protease, and finally screening 1 strain with obviously improved alkaline protease activity.
3.2 Shake flask rescreening
Inoculating the recombinant strain into 5mL of LB liquid culture medium (containing kanamycin and 50 mug/mL), culturing at 37 ℃ and 220r/min overnight, transferring into 50mL of fresh LB culture medium according to the inoculum size of 2%, continuously culturing at 37 ℃ and 220r/min for 48h, taking the supernatant as a crude enzyme solution, and measuring the alkaline protease activity of the obtained enzyme solution to obtain the strain with remarkably improved alkaline protease activity.
Example 4: determination of sequences of alkaline protease mutants
The alkaline protease gene sequence is extracted from the strain and sequenced (Beijing Hua big bioengineering company), and the result shows that the nucleotide sequence of the alkaline protease mutant gene obtained by amplification is shown as SEQ ID NO: 5, the coding gene was designated as prom.
Example 5: investigation of enzymatic Properties of alkaline protease mutants
The crude alkaline protease solutions obtained in examples 1 and 3 were salted out using 70% saturated ammonium sulfate. The precipitate formed was dialyzed against MES buffer (20mmol/L, pH 7.0; buffer A), the retentate was subjected to ion exchange chromatography on a CM-Sephadex column (2.5X 20CM) pre-equilibrated with buffer A, and the protein was eluted with a linear gradient using buffer A containing 0 to 1mol/L NaCl. The eluate containing protease activity was applied to a Superdex G-75 gel column (1.6X 80cm) pre-equilibrated with buffer A. The purified protein was then eluted with buffer A (0.5 ml/min). And (3) freeze-drying the purified enzyme solution to prepare alkaline protease enzyme powder, and dissolving the alkaline protease enzyme powder in purified water for enzyme property investigation.
Enzyme activity of alkaline protease: the enzyme activity of alkaline protease (shown in figure 1) was measured at 40 ℃ and pH 10, and the enzyme activities of CPR, LPR and PROM were 15.63U/mL, 46.87U/mL and 61.75U/mL, respectively.
Optimum temperature for alkaline protease mutants: the enzymatic activity was measured at pH 10 between 30-80 c (as shown in fig. 2) and the temperature optimum for PROM was 60 c.
Optimum pH of alkaline protease mutant: the enzymatic activity was measured at a temperature of 60 ℃ at pH 7.0-12.0 (as shown in fig. 3), and the pH optimum of PROM was 10.0.
Thermostability of alkaline protease mutants: the residual enzyme activity after 20-60 ℃ incubation was measured at pH 10 (as shown in fig. 4), and after 20h incubation at 20 ℃, 30 ℃, 40 ℃, 50 ℃ and 60 ℃ for 20h, the PROM residual enzyme activity was 96.69%, 86.02%, 80.42%, 67.47% and 53.27%, respectively.
pH stability of alkaline protease mutants: the residual enzyme activity after 20h incubation at pH 7.0-11.0 was determined at 40 ℃ (as shown in fig. 5), and after 20h incubation at pH7.0, 8.0, 9.0, 10.0 and 11.0, the PROM residual enzyme activity was 74.39%, 81.42%, 85.43%, 80.43% and 63.92%.
Although the present invention has been disclosed in the form of preferred embodiments, it is not intended to limit the present invention, and those skilled in the art may make various changes, modifications, substitutions and alterations in form and detail without departing from the spirit and principle of the present invention, the scope of which is defined by the appended claims and their equivalents.
Sequence listing
<110> Tianjin science and technology university, Shandong Longkote enzyme preparations Co., Ltd
<120> an alkaline protease mutant and use thereof
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Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly Ile
100 105 110
Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly Ala
115 120 125
Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His Pro
130 135 140
Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala Tyr
145 150 155 160
Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val Ala
165 170 175
Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val Ser
180 185 190
Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr Ser
195 200 205
Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp Val
210 215 220
Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys Gln
225 230 235 240
Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala Ala
245 250 255
Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro Ala
260 265 270
Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser Asn
275 280 285
Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala Pro
290 295 300
Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr Leu
305 310 315 320
Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala Leu
325 330 335
Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn Arg
340 345 350
Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly Lys
355 360 365
Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 5
<211> 1409
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gctcaaccgg cgaaaaatgt tgaaaaggat tatattgtcg gatttaagtc aggagtgaaa 60
accgcatctg tcaaaaagga catcatcaaa gagagcggcg gaaaagtgga caagcagttt 120
agaatcatca acgcggcaaa agcgaagcta gacaaagaag cgcttaagga agtcaaaaat 180
gatccggatg tcgcttatgt ggaagaggat catgtggccc atgccttggc gcaaaccgtt 240
ccttacggca ttcctctcat taaagcggac aaagtgcagg ctcaaggctt taagggagcg 300
aatgtaaaag tagccgtcct ggatacagga atccaagcct ctcatccgga cttgaacgta 360
gtcggcggag caagttttgt ggctggcgag gcttataaca ccgacggcaa cggacacggc 420
acacatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaactcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tagacaatgc atatgcaaga 660
ggggttgtcg ttgtagcagc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggcg cggtagactc taacagcaac 780
agagcttcat tttccagtgt gggagcagag cttgaagtca tggctcctgg cgcaggcgta 840
tacagcactt acccaacgaa cacttatgca acattgaacg gaacgtcaat ggcttctcct 900
catgtagcgg gagcagcagc tttgatcttg tcaaaacatc cgaacctttc agcttcacaa 960
gtccgcaacc gtctctccag cacggcgact tatttgggaa gctccttcta ctatgggaaa 1020
ggtctgatca atgtcgaagc tgccgctcaa taacgaattc aggtgcaggc tcaatcagct 1080
atccggcccg ttatgcgaac gcaatggcag tcggagctac tgaccaaaac aacaaccgcg 1140
ccagcttttc acagtatggc gcagggcttg acattgtcgc accaggtgta aacgtgcaga 1200
gcacataccc aggttcaacg tatgccagct taaacggtac atcgatggct actcctcatg 1260
ttgcaggtgc agcagccctt gttaaacaaa agaacccatc ttggtccaat gtacaaatcc 1320
gcaatcatct aaagaatacg gcaacgagct taggaagcac gaacttgtat ggaagcggac 1380
ttgtcaatgc agaagcggca acacgctaa 1409
<210> 6
<211> 451
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Ala Gln Pro Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys
1 5 10 15
Ser Gly Val Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser
20 25 30
Gly Gly Lys Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala
35 40 45
Lys Leu Asp Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val
50 55 60
Ala Tyr Val Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val
65 70 75 80
Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly
85 90 95
Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln
100 105 110
Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala
115 120 125
Gly Glu Ala Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala
130 135 140
Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala
145 150 155 160
Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser
165 170 175
Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn
180 185 190
Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr
195 200 205
Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val
210 215 220
Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile
225 230 235 240
Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp
245 250 255
Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu
260 265 270
Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr
275 280 285
Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
290 295 300
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln
305 310 315 320
Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe
325 330 335
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln Met Ala
340 345 350
Val Gly Ala Thr Asp Gln Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr
355 360 365
Gly Ala Gly Leu Asp Ile Val Ala Pro Gly Val Asn Val Gln Ser Thr
370 375 380
Tyr Pro Gly Ser Thr Tyr Ala Ser Leu Asn Gly Thr Ser Met Ala Thr
385 390 395 400
Pro His Val Ala Gly Ala Ala Ala Leu Val Lys Gln Lys Asn Pro Ser
405 410 415
Trp Ser Asn Val Gln Ile Arg Asn His Leu Lys Asn Thr Ala Thr Ser
420 425 430
Leu Gly Ser Thr Asn Leu Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala
435 440 445
Ala Thr Arg
450
Claims (9)
1. An alkaline protease mutant, wherein the amino acid sequence of the alkaline protease mutant is as shown in SEQ ID NO: and 6.
2. A gene encoding the alkaline protease mutant according to claim 1.
3. A recombinant vector comprising the gene of claim 2.
4. A host cell comprising the gene of claim 2 or the recombinant vector of claim 3.
5. The recombinant vector of claim 3, wherein the vector is pWB 980.
6. The host cell of claim 4, wherein the host cell is Bacillus subtilis WB 600.
7. Use of the alkaline protease mutant according to claim 1 for hydrolyzing peptide bonds of proteins to produce polypeptides or amino acids.
8. The use of claim 7, wherein the mutant has increased alkaline protease activity compared to the wild type.
9. The method for preparing the alkaline protease mutant according to claim 1, comprising the steps of:
(1) the peptide as shown in SEQ ID NO: 5, performing double enzyme digestion and connection on the alkaline protease mutant gene shown in the 5 and the linearized pWB980 vector through HindIII and BamHI to obtain a recombinant vector;
(2) transforming the recombinant vector into bacillus subtilis WB600 to obtain a recombinant strain;
(3) expressing the recombinant strain, and purifying the expression product to obtain the alkaline protease mutant.
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| CN202210097345.8A CN114480352B (en) | 2021-01-28 | 2022-01-26 | Alkaline protease mutant and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112725316A (en) * | 2021-03-04 | 2021-04-30 | 湖南夏盛酶技术有限公司 | Alkallikrein 2018 mutant and preparation method thereof |
| CN114480352A (en) * | 2021-01-28 | 2022-05-13 | 天津科技大学 | Alkaline protease mutant and application thereof |
| CN114807100A (en) * | 2022-04-28 | 2022-07-29 | 湖北大学 | Alkaline protease gene sequence suitable for bacillus licheniformis expression and application thereof |
| CN118147117A (en) * | 2024-05-11 | 2024-06-07 | 中国农业科学院生物技术研究所 | Heat-resistant feeding protease mutant and application thereof |
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| WO2004058961A1 (en) * | 2002-12-20 | 2004-07-15 | Henkel Kommanditgesellschaft Auf Aktien | Subtilisin variants, with improved perhydrolase activity |
| CN110862979A (en) * | 2020-01-20 | 2020-03-06 | 中国科学院天津工业生物技术研究所 | Mutant of alkaline protease and application thereof |
| CN111334494A (en) * | 2019-10-09 | 2020-06-26 | 天津科技大学 | Screening of novel high-stability alkaline protease mutants |
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| CN110923222B (en) * | 2019-12-13 | 2021-11-23 | 中国科学院天津工业生物技术研究所 | Novel alkaline protease acid mutant from bacillus licheniformis |
| CN112662654A (en) * | 2021-01-28 | 2021-04-16 | 天津科技大学 | Alkaline protease mutant and application thereof |
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| WO2004058961A1 (en) * | 2002-12-20 | 2004-07-15 | Henkel Kommanditgesellschaft Auf Aktien | Subtilisin variants, with improved perhydrolase activity |
| CN111334494A (en) * | 2019-10-09 | 2020-06-26 | 天津科技大学 | Screening of novel high-stability alkaline protease mutants |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480352A (en) * | 2021-01-28 | 2022-05-13 | 天津科技大学 | Alkaline protease mutant and application thereof |
| CN112725316A (en) * | 2021-03-04 | 2021-04-30 | 湖南夏盛酶技术有限公司 | Alkallikrein 2018 mutant and preparation method thereof |
| CN112725316B (en) * | 2021-03-04 | 2022-09-06 | 宁夏夏盛实业集团有限公司 | Alkallikrein 2018 mutant and preparation method thereof |
| CN114807100A (en) * | 2022-04-28 | 2022-07-29 | 湖北大学 | Alkaline protease gene sequence suitable for bacillus licheniformis expression and application thereof |
| CN114807100B (en) * | 2022-04-28 | 2023-06-27 | 湖北大学 | Alkaline protease gene sequence suitable for bacillus licheniformis expression and application |
| CN118147117A (en) * | 2024-05-11 | 2024-06-07 | 中国农业科学院生物技术研究所 | Heat-resistant feeding protease mutant and application thereof |
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| CN114480352A (en) | 2022-05-13 |
| CN114480352B (en) | 2023-07-04 |
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