CN108004220B - Alkaline protease BmP mutant for improving thermal stability and gene and application thereof - Google Patents

Alkaline protease BmP mutant for improving thermal stability and gene and application thereof Download PDF

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CN108004220B
CN108004220B CN201711414723.6A CN201711414723A CN108004220B CN 108004220 B CN108004220 B CN 108004220B CN 201711414723 A CN201711414723 A CN 201711414723A CN 108004220 B CN108004220 B CN 108004220B
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刘丹妮
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    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus

Abstract

The invention relates to the field of bioengineering, in particular to an alkaline protease BmP mutant for improving thermal stability and a gene and application thereof. The mutant is obtained by mutating 193 th position of alkaline protease BmP mutant with amino acid sequence shown as SEQ ID NO.6 from amino acid S to L or K; alternatively, 288 is mutated from amino acid S to K or M. The invention improves the thermal stability of the Bacillus mojavensis (Bacillus mojavensis) alkaline protease BmP, obviously improves the thermal stability of the enzyme on the premise of not damaging other excellent enzymological properties of the enzyme, and lays a foundation for the industrial application of the enzyme.

Description

Alkaline protease BmP mutant for improving thermal stability and gene and application thereof
Technical Field
The invention relates to the field of bioengineering, in particular to an alkaline protease BmP mutant for improving thermal stability and a gene and application thereof.
Background
Protease is a kind of enzyme catalyzing protein hydrolysis, and the protease is widely applied to the fields of feed processing, brewing, washing, food processing and the like as an important industrial enzyme preparation. The protease is widely distributed, the protease on the market at present mainly comes from microorganisms, and compared with animals and plants, the microbial protease has wide pH value range and low production cost. Bacillus proteases are the most important class of microbial proteases, mainly classified into neutral proteases and alkaline proteases, and currently, the Bacillus proteases are widely used in the fields of feeds, washing, foods and the like.
Bacillus mojavensis (Bacillus mojavensis) alkaline protease BmP for short, and experiments show that the Bacillus mojavensis alkaline protease has strong capability of decomposing protein raw materials in the early stage, and is suitable for industrial fields of food, feed and the like. However, BmP has poor thermal stability and is easy to decompose and deactivate at a high temperature of more than 70 ℃, thereby limiting the industrial application of the BmP. Therefore, the heat stability of the BmP is improved, and the problem to be solved is urgently needed in the industrial application of the BmP.
Disclosure of Invention
The invention carries out protein molecule modification on the BmP which is derived from Bacillus mojavensis alkaline protease, thereby improving the stability of the BmP in a high-temperature environment and laying a foundation for the industrial application of the BmP.
The invention aims to provide a mutant of alkaline protease BmP with improved heat stability.
Still another object of the present invention is to provide a gene encoding a BmP mutant of alkaline protease having improved thermostability.
The nucleotide sequence of the alkaline protease BmP of the bacillus mojavensis is shown as SEQ ID NO. 1.
SEQ ID NO.1
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTCGGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGGCAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCATGTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCGGAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCTGCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTATCCTTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGAGCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTAGCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGTAGACTCTAACAGCAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
According to the specific embodiment of the invention, the method of site-directed saturation mutagenesis is adopted to mutate 193 rd and 288 th positions of alkaline protease BmP with an amino acid sequence shown as SEQ ID NO.6, and effective mutated amino acids of 2 sites of 193 rd position and 288 th position are determined through screening. Wherein the effective mutation amino acid at position 193 is L or K, preferably L; the 288 significant mutation amino acid is M or K, wherein M is the best.
On the basis of effective mutation sites, four alkaline protease BmP mutants with improved thermal stability are obtained finally and are named as BmP1, BmP2, BmP3 and BmP4 respectively. The retention of BmP1, BmP2, BmP3 and BmP4 under water bath treatment at 75 ℃ for 5 minutes was 65%, 60%, 56% and 49%, respectively, whereas the retention of the original enzyme BmP under the same conditions was only 9%. BmP1 contains mutation sites S193K and S288M; BmP2 contains mutation sites S193L and S288M; BmP3 contains mutation sites S193L and S288K; BmP4 contains mutation sites of S193L and S288K, nucleotide sequences of BmP1, BmP2, BmP3 and BmP4 mutants are shown in SEQ ID NO.2 to SEQ ID NO.5, and amino acid sequences are shown in SEQ ID NO.7 to SEQ ID NO. 10.
SEQ ID NO.2 (mutant BmP1)
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTCGGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGGCAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCATGTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCGGAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCTGCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTAAAATTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGAGCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTAGCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGTAGACTCTAACATGAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
Nucleotide sequence SEQ ID No.3 (mutant BmP 2):
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTCGGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGGCAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCATGTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCGGAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCTGCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTACTCTTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGAGCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTAGCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGTAGACTCTAACATGAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
nucleotide sequence SEQ ID No.4 (mutant BmP 3):
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTCGGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGGCAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCATGTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCGGAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCTGCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTACTCTTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGAGCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTAGCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGTAGACTCTAACAAAAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
nucleotide sequence SEQ ID No.5 (mutant BmP 4):
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTCGGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGGCAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCATGTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCGGAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCTGCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTAAAATTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGAGCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTAGCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGTAGACTCTAACAAAAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
amino acid sequence SEQ ID NO.6 (original alkaline protease BmP):
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
amino acid sequence SEQ ID NO.7 (mutant BmP1)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVKLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNMNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
Amino acid sequence SEQ ID NO.8 (mutant BmP2)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVLLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNMNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
Amino acid sequence SEQ ID NO.9 (mutant BmP3)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVLLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNKNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
Amino acid sequence SEQ ID NO.10 (mutant BmP4)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVKLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNKNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
The invention also provides a recombinant vector containing a gene coding the alkaline protease BmP mutant with improved thermal stability.
The invention also provides a host cell containing the gene for encoding the BmP mutant of the alkaline protease with improved thermal stability.
The method for obtaining the alkaline protease BmP mutant with improved thermal stability comprises the steps of mutating the 193 th position of the alkaline protease BmP mutant with an amino acid sequence shown as SEQ ID NO.6 from amino acid S to L or K; alternatively, 288 is a step in which amino acid S is mutated to K or M.
The invention also provides application of the alkaline protease BmP mutant for improving the thermal stability.
The method for preparing the alkaline protease BmP mutant with improved heat stability by fermentation according to the invention comprises the step of fermentation by using the recombinant vector.
The invention improves the thermal stability of the Bacillus mojavensis (Bacillus mojavensis) alkaline protease BmP, obviously improves the thermal stability of the enzyme on the premise of not damaging other excellent enzymological properties of the enzyme, and lays a foundation for the industrial application of the enzyme.
Drawings
FIG. 1 shows the optimal reaction pH for the original alkaline protease BmP and the mutants BmP1 to BmP 4;
FIG. 2 shows the pH stability of the original alkaline protease BmP and the mutant BmP1 to BmP 4;
FIG. 3 shows the optimal reaction temperatures for the original alkaline protease BmP and the mutants BmP1 to BmP 4.
Detailed Description
The molecular biology experiments, which are not specifically described in the following examples, were performed according to the specific methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions; the reagents and biomaterials, if not specifically indicated, are commercially available.
Experimental materials and reagents:
1. bacterial strains and vectors
Escherichia coli strain Topl0, Bacillus subtilis WB600 and expression vector phyP43L (Bacillus subtilis P43 promoter and alkaline protease signal peptide are connected to the vector pHY300PLK to obtain expression vector phyP43L)。
2. Enzyme and kit
Q5 high fidelity Taq enzyme MIX was purchased from NEB company, plasmid extraction, gel purification, restriction enzyme, kit was purchased from Shanghai Biotech company.
3. Culture medium
The E.coli medium was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). The Bacillus subtilis culture medium is LBK, namely LB culture medium plus kanamycin.
Example 1 cloning of Bacillus mojavensis (Bacillus mojavensis) alkaline protease BmP Gene
The target gene was directly synthesized based on the reported sequence of Bacillus mojavensis alkaline protease gene (Genebank: AY 665611.1). Two primers (R: 5'-ATCGGGATCCGCTCAACCGGCGAAAAATGTT-3' and F: 5'-TCTAGCGGCCGC TTATTGAGCGGCAGCTTCGAC-3') were designed based on the synthesized target gene for amplification of the BmP gene of Bacillus mojavensis alkaline protease. Purifying and recovering the amplified PCR product, and connecting the PCR product to an expression vector phyP43L, obtaining an expression vector phyP43L-BmP。
Example 2 site-directed saturation mutagenesis
The effect of position 193 and 288 on the thermostability of the alkaline protease BmP was investigated by saturation mutagenesis. The process of site-directed saturation mutagenesis is as follows: to construct a good phyP43Performing PCR amplification by using the corresponding mutation primer by using L-BmP as a template; and (4) carrying out agarose electrophoresis on the amplified PCR product, and purifying and recovering the PCR product. Decomposing the original plasmid by using restriction endonuclease DpnI, transferring the decomposed product into escherichia coli Top10 by using a heat shock method, verifying a recombinant transformant by using a bacterial liquid PCR, extracting a plasmid of the transformant which is verified to be correct, and sequencing to determine a corresponding mutant. The correctly sequenced mutants were transformed into Bacillus subtilis WB600 by electrotransformation.
Recombinant transformants were screened as follows: firstly, inoculating recombinant bacteria growing on a kanamycin resistant plate into an LBK culture medium, and culturing for 24 hours at 37 ℃ and 200 rpm; centrifuging the cultured bacterial liquid, taking the supernatant, and performing enzyme activity determination by referring to national standard 2009; the thermostability of the mutants was determined at 75 ℃ for 5 minutes in a water bath. The results of the experiment are shown in table 1, and it can be seen from table 1 that: after 193-position saturation mutation, two mutant amino acids are screened, so that the heat stability of BmP can be improved, wherein the two mutant amino acids are S193L and S193K respectively, and the retention rates are 30% and 28% respectively; two mutant amino acids are also screened at position 288, which can effectively improve the heat stability of BmP and are respectively S288M and S288K, and the retention rate is respectively 41 percent and 38 percent
TABLE 1 thermostability of the original alkaline protease BmP and mutants
Numbering Retention (%)
Original alkaline protease BmP 8
S193L 30
S193K 28
S288M 41
S288K 38
Example 3 combinatorial mutagenesis
The combined mutation is carried out on the basis of single point mutation, and 4 combined mutations are finally obtained through experiments and are named as BmP1, BmP2, BmP3 and BmP4 respectively. Wherein BmP1 comprises mutation sites of S193K and S288M; BmP2 contains mutation sites S193L and S288M; BmP3 contains mutation sites S193L and S288K; BmP3 contains mutation sites S193K and S288K.
Example 4 thermostability assay of the original alkaline protease BmP and mutants
In order to compare the thermostability of the original alkaline protease BmP and the mutant accurately, the corresponding protease was first purified by nickel column purification. The purified alkaline protease BmP and the mutant were subjected to a water bath treatment at 75 ℃ for 5 minutes to determine thermal stability. The retention of mutants BmP1, BmP2, BmP3 and BmP4 under these conditions was finally determined by experiments to be 65%, 60%, 56% and 49%, respectively, while the original alkaline protease BmP was 9%.
TABLE 2 original alkaline protease BmP and combinatorial mutant thermostability
Numbering Retention (%)
Original alkaline protease BmP 9
BmP1：S193K/S288M 65
BmP2：S193L/S288M 60
BmP3：S193L/S288K 56
BmP3：S193K/S288K 49
Example 5 optimal reaction pH and pH stability of original alkaline protease BmP and mutant
The optimum reaction pH of the original alkaline protease BmP and the mutants BmP1, BmP2, BmP3 and BmP4 were determined by reference to the national standard method. The optimal reaction pH for the original alkaline protease BmP and the mutants BmP1, BmP2, BmP3 and BmP4 are shown in FIG. 1. As can be seen from FIG. 1, the optimum pH values of the mutants BmP1, BmP2, BmP3 and BmP4 are almost the same as that of the original alkaline protease BmP, and are all 10.0.
The original alkaline protease BmP and the mutants BmP1, BmP2, BmP3 and BmP4 are respectively treated for 2 hours at room temperature under the condition of pH6-11, and then the enzyme activity is determined by the method of national standard, and the result is shown in figure 2. From FIG. 2, it can be seen that the pH stability of the mutants BmP1, BmP2, BmP3 and BmP4 is consistent with that of the original alkaline protease BmP.
Example 6 optimum reaction temperature for alkaline protease BmP and mutant
The optimum reaction temperatures of the original alkaline protease BmP and the mutants BmP1, BmP2, BmP3 and BmP4 were determined by reference to the national standard method, and the results are shown in FIG. 3. As can be seen from FIG. 3, the optimum reaction temperature for the alkaline protease BmP was 65 ℃ and for the mutants BmP1, BmP2, BmP3 and BmP4 was 70 ℃.
Sequence listing
<110> Liudanni
<120> alkaline protease BmP mutant for improving heat stability and gene and application thereof
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1140
<212> DNA
<213> Bacillus mojavensis (Bacillus mojavensis)
<400> 1
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60
atggcattcg gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat 120
attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcacg catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtatcct tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggtgcgg tagactctaa cagcaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
<210> 2
<211> 1140
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60
atggcattcg gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat 120
attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcacg catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtaaaat tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggtgcgg tagactctaa catgaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
<210> 3
<211> 1140
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60
atggcattcg gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat 120
attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcacg catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtactct tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggtgcgg tagactctaa catgaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
<210> 4
<211> 1140
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60
atggcattcg gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat 120
attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcacg catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtactct tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggtgcgg tagactctaa caaaaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
<210> 5
<211> 1140
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg 60
atggcattcg gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat 120
attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag 180
agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac 240
aaagaagcgc ttaaggaagt caaaaatgat ccggatgtcg cttatgtgga agaggatcat 300
gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa 360
gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc 420
caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct 480
tataacaccg acggcaacgg acacggcacg catgttgccg gtacagtagc tgcgcttgac 540
aatacaacgg gtgtattagg cgttgcgcca agcgtaaaat tgtacgcggt taaagtactg 600
aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca 660
aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa 720
caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc 780
ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct 840
gttggtgcgg tagactctaa caaaaacaga gcttcatttt ccagtgtggg agcagagctt 900
gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca 960
ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca 1020
aaacatccga acctttcagc ttcacaagtc cgcaaccgtc tctccagcac ggcgacttat 1080
ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaagctgc cgctcaataa 1140
<210> 6
<211> 379
<212> PRT
<213> Bacillus mojavensis (Bacillus mojavensis)
<400> 6
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 Gly 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 Ser 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 Ala 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 Asn 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> 7
<211> 379
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
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 Gly 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
Lys Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser 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 Ala 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 Met
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 Asn 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> 8
<211> 379
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
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 Gly 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
Leu Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser 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 Ala 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 Met
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 Asn 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> 9
<211> 379
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
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 Gly 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
Leu Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser 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 Ala 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 Lys
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 Asn 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> 10
<211> 379
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
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 Gly 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
Lys Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser 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 Ala 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 Lys
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 Asn 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

Claims (7)

1. The alkaline protease BmP mutant for improving the thermal stability is characterized in that the 193 th site of the alkaline protease BmP mutant with the amino acid sequence shown as SEQ ID NO.6 is mutated from amino acid S to L or K; alternatively, 288 is mutated from amino acid S to K or M.
2. A gene encoding the BmP mutant of alkaline protease with improved thermostability according to claim 1.
3. A recombinant vector comprising the gene of claim 2.
4. A host cell comprising the gene of claim 2.
5. A method for obtaining an alkaline protease BmP mutant with improved heat stability is characterized by comprising the steps of mutating 193 th site of the alkaline protease BmP mutant with an amino acid sequence shown as SEQ ID NO.6 from amino acid S to L or K; or, the 288 th amino acid S is mutated to K or M.
6. The use of the BmP mutant of alkaline protease with improved thermal stability as claimed in claim 1 for catalyzing proteolysis.
7. A method for preparing an alkaline protease BmP mutant with improved thermal stability by fermentation, which comprises the step of performing fermentation by using the recombinant vector of claim 3.
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CN108570461B (en) * 2018-04-17 2020-08-11 横琴仲泰生物医药有限公司 Alkaline protease BmP mutant for improving specific activity and coding gene thereof
CN110923221B (en) * 2019-12-13 2021-10-22 中国科学院天津工业生物技术研究所 Alkaline protease high-temperature mutant from bacillus licheniformis
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
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CN1113953A (en) * 1994-06-01 1995-12-27 中国科学院生物物理研究所 No.118 Position mutation of bacillus subtilis alkaline protease and its thermal stable enzyme
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HU231053B1 (en) * 2011-09-08 2020-03-30 Szegedi Tudományegyetem Copper-resistant, fengycin hyperproducing bacillus mojavensis strain for protection against plant pests, its use and compounds containing the same
CN102936588B (en) * 2012-12-10 2014-01-08 江南大学 Protease with improved thermal stability as well as construction method and application thereof
CN103436511B (en) * 2013-06-28 2015-03-04 国家海洋局第三海洋研究所 High temperature alkaline protease and preparation method thereof
CN104017791B (en) * 2014-06-19 2017-02-15 江南大学 Keratinase mutant with improved thermal stability and preparation method thereof
CA2963148A1 (en) * 2014-10-28 2016-05-06 Agrivida, Inc. Methods and compositions for stabilizing trans-splicing intein modified proteases

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