CN108004220A - Improve alkali protease BmP mutant and its gene and the application of heat endurance - Google Patents

Improve alkali protease BmP mutant and its gene and the application of heat endurance Download PDF

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CN108004220A
CN108004220A CN201711414723.6A CN201711414723A CN108004220A CN 108004220 A CN108004220 A CN 108004220A CN 201711414723 A CN201711414723 A CN 201711414723A CN 108004220 A CN108004220 A CN 108004220A
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CN108004220B (en
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刘丹妮
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • 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 present invention relates to bioengineering field, and in particular to improves alkali protease BmP mutant and its gene and the application of heat endurance.The mutant is 193 of the mutant of the alkali protease BmP by amino acid sequence as shown in SEQ ID NO.6 and sports L or K by amino acid S；Alternatively, 288 is sport K or M is obtained by amino acid S.The present invention improves the heat endurance of Mo Haiwei bacillus (Bacillus mojavensis) alkali protease BmP, on the premise of the enzyme other excellent zymologic properties are not destroyed, the heat endurance of the enzyme is significantly improved, is laid the foundation for its commercial application.

Description

Improve alkali protease BmP mutant and its gene and the application of heat endurance
Technical field
The present invention relates to bioengineering field, and in particular to improve heat endurance alkali protease BmP mutant and its Gene and application.
Background technology
Protease is the class of enzymes of catalytic proteins hydrolysis, and protease widely should as a kind of important industrial enzyme preparation For fields such as feed processing, brewing, washing, food processings.Protease is widely distributed, and protease on the market mainly comes at present Microorganism is come from, relative to animal and plant, microbial protease pH value range is wide, and production cost is low.Bacillus albumen Enzyme is broadly divided into neutral proteinase and alkali protease, at present bacillus as the most important one kind of microbial protease Protease has been widely used in the fields such as feed, washing, food.
Mo Haiwei bacillus (Bacillus mojavensis) alkali protease abbreviation BmP, is found through experiments that early period It is strong to protein raw materials capacity of decomposition, suitable for industrial circles such as food, feeds.But BmP heat endurances are poor, more than 70 DEG C At a high temperature of easily decompose inactivation, limit its commercial application.Therefore the heat endurance of BmP is improved, is BmP commercial applications Urgent problem.
The content of the invention
The present invention from Mo Haiwei bacillus (Bacillus mojavensis) alkali protease BmP to carrying out egg White matter molecular modification, so as to improve the stability of BmP in high temperature environments, lays the foundation for the commercial application of BmP.
The object of the present invention is to provide the mutant for improving heat endurance alkali protease BmP.
Another object of the present invention is to provide the encoding gene for improving heat endurance alkali protease BmP mutant.
The nucleotide sequence of the alkali protease BmP of Mo Haiwei bacillus such as SEQ ID NO.1.
SEQ ID NO.1
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTC GGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAA AACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGG CAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCAT GTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTT TAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCG GAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCT GCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTATCCTTGTACGCGGTTAAAGTACTGAATTCAAG CGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGA GCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTA GCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGC TGTTGGTGCGGTAGACTCTAACAGCAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTG GCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTA GCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCAC GGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
Embodiment according to the present invention, the present invention using fixed point saturation mutation method to amino acid sequence such as The 193rd and the 288th of alkali protease BmP shown in SEQ ID NO.6 is mutated, and the 193rd is determined by screening Position and effective mutating acid in the 288th this 2 sites.Wherein effective mutating acid of 193 is respectively L or K, wherein L is best；288 effective mutating acids are respectively M or K, and wherein M is best.
On the basis of effective mutational site, it is respectively combined, has finally obtained the alkalescence that four heat endurances improve Cathepsin B mP mutant, is respectively designated as BmP1, BmP2, BmP3 and BmP4.At 75 DEG C, water bath processing is under the conditions of 5 minutes, The retention rate of BmP1, BmP2, BmP3 and BmP4 are respectively 65%, 60%, 56% and 49%, and protoenzyme BmP is in the same terms Under retention rate be only 9%.The mutational site that BmP1 is included is S193K and S288M；The mutational site that BmP2 is included is S193L And S288M；The mutational site that BmP3 is included is S193L and S288K；The mutational site that BmP4 is included is S193L and S288K, The nucleotide sequence of BmP1, BmP2, BmP3 and BmP4 mutant is as shown in SEQ ID NO.2 to SEQ ID NO.5, amino acid sequence Row are as shown in SEQ ID NO.7 to SEQ ID NO.10.
SEQ ID NO.2 (mutant BmP1)
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTC GGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAA AACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGG CAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCAT GTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTT TAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCG GAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCT GCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTAAAATTGTACGCGGTTAAAGTACTGAATTCAAG CGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGA GCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTA GCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGC TGTTGGTGCGGTAGACTCTAACATGAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTG GCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTA GCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCAC GGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
Nucleotide sequence SEQ ID NO.3 (mutant BmP2)：
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTC GGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAA AACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGG CAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCAT GTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTT TAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCG GAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCT GCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTACTCTTGTACGCGGTTAAAGTACTGAATTCAAG CGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGA GCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTA GCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGC TGTTGGTGCGGTAGACTCTAACATGAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTG GCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTA GCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCAC GGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
Nucleotide sequence SEQ ID NO.4 (mutant BmP3)：
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTC GGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAA AACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGG CAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCAT GTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTT TAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCG GAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCT GCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTACTCTTGTACGCGGTTAAAGTACTGAATTCAAG CGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGA GCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTA GCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGC TGTTGGTGCGGTAGACTCTAACAAAAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTG GCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTA GCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCAC GGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
Nucleotide sequence SEQ ID NO.5 (mutant BmP4)：
ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTCATGCTCGTGTTCACGATGGCATTC GGCGATTCCGCTTCTGCTGCTCAACCGGCGAAAAATGTTGAAAAGGATTATATTGTCGGATTTAAGTCAGGAGTGAA AACCGCATCTGTCAAAAAGGACATCATCAAAGAGAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGG CAAAAGCGAAGCTAGACAAAGAAGCGCTTAAGGAAGTCAAAAATGATCCGGATGTCGCTTATGTGGAAGAGGATCAT GTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTCCTCTCATTAAAGCGGACAAAGTGCAGGCTCAAGGCTT TAAGGGAGCGAATGTAAAAGTAGCCGTCCTGGATACAGGAATCCAAGCTTCTCATCCGGACTTGAACGTAGTCGGCG GAGCAAGCTTTGTGGCTGGCGAAGCTTATAACACCGACGGCAACGGACACGGCACGCATGTTGCCGGTACAGTAGCT GCGCTTGACAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTAAAATTGTACGCGGTTAAAGTACTGAATTCAAG CGGAAGCGGATCATACAGCGGCATTGTAAGCGGAATCGAGTGGGCGACAACAAACGGCATGGATGTTATCAATATGA GCCTTGGGGGAGCATCAGGCTCGACAGCGATGAAACAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTA GCTGCAGCAGGGAACAGCGGATCTTCAGGAAACACGAATACAATTGGCTATCCTGCGAAATACGATTCTGTCATCGC TGTTGGTGCGGTAGACTCTAACAAAAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTG GCGCAGGCGTATACAGCACTTACCCAACGAACACTTATGCAACATTGAACGGAACGTCAATGGCTTCTCCTCATGTA GCGGGAGCAGCAGCTTTGATCTTGTCAAAACATCCGAACCTTTCAGCTTCACAAGTCCGCAACCGTCTCTCCAGCAC GGCGACTTATTTGGGAAGCTCCTTCTACTATGGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA
Amino acid sequence SEQ ID NO.6 (original alkaline Cathepsin B mP)：
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQF RIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPD LNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGM DVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAEL EVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAA AQ
Amino acid sequence SEQ ID NO.7 (mutant BmP1)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQF RIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPD LNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVKLYAVKVLNSSGSGSYSGIVSGIEWATTNGM DVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNMNRASFSSVGAEL EVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAA AQ
Amino acid sequence SEQ ID NO.8 (mutant BmP2)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQF RIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPD LNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVLLYAVKVLNSSGSGSYSGIVSGIEWATTNGM DVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNMNRASFSSVGAEL EVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAA AQ
Amino acid sequence SEQ ID NO.9 (mutant BmP3)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQF RIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPD LNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVLLYAVKVLNSSGSGSYSGIVSGIEWATTNGM DVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNKNRASFSSVGAEL EVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAA AQ
Amino acid sequence SEQ ID NO.10 (mutant BmP4)
MMRKKSFWLGMLTAFMLVFTMAFGDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQF RIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPD LNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVKLYAVKVLNSSGSGSYSGIVSGIEWATTNGM DVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNKNRASFSSVGAEL EVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAA AQ
Present invention also offers the weight of the gene comprising the alkali protease BmP mutant for encoding above-mentioned raising heat endurance Group carrier.
Present invention also offers the place of the gene comprising the alkali protease BmP mutant for encoding above-mentioned raising heat endurance Chief cell.
Obtaining the side for the alkali protease BmP mutant for improving heat endurance according to the present invention is included amino acid sequence 193 of the mutant of alkali protease BmP as shown in SEQ ID NO.6 sport L or K by amino acid S；Alternatively, 288 are The step of K or M is sported by amino acid S.
Present invention also offers the application of the alkali protease BmP mutant of above-mentioned raising heat endurance.
The method for the alkali protease BmP mutant that fermentation preparation improves heat endurance includes the use of according to the present invention The step of recombinant vector stated is fermented.
The present invention improves the thermostabilization of Mo Haiwei bacillus (Bacillus mojavensis) alkali protease BmP Property, on the premise of the enzyme other excellent zymologic properties are not destroyed, the heat endurance of the enzyme is significantly improved, should for its industrialization With laying the foundation.
Brief description of the drawings
Fig. 1 shows the optimal reaction pH of original alkaline Cathepsin B mP and mutant BmP1 to BmP4；
Fig. 2 shows the pH stability of original alkaline Cathepsin B mP and mutant BmP1 to BmP4；
Fig. 3 shows the optimal reactive temperature of original alkaline Cathepsin B mP and mutant BmP1 to BmP4.
Embodiment
Do not make the experimental methods of molecular biology illustrated, equal reference in following embodiments《Molecular Cloning:A Laboratory guide》 Listed specific method carries out in one book of (third edition) J. Pehanorm Brookers, or is carried out according to kit and product description； The reagent and biomaterial, unless otherwise specified, commercially obtain.
Experiment material and reagent：
1st, bacterial strain and carrier
Coli strain Topl0, bacillus subtilis WB600, expression vector phyP43L is (by bacillus subtilis P43 Promoter and alkaline protease signal peptide are connected to carrier pHY300PLK and obtain expression vector phyP43L)。
2nd, enzyme and kit
Q5 high-fidelity Taq enzymes MIX is purchased from NEB companies, and plasmid extraction, glue purification, restriction enzyme, kit are purchased from upper Hai Shenggong companies.
3rd, culture medium
Escherichia coli culture medium is LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).Withered grass gemma Culture medium is LBK, i.e. LB culture mediums add kanamycins.
The clone of embodiment 1, Mo Haiwei bacillus (Bacillus mojavensis) alkali protease BmP genes
According to the sequence (Genebank for having reported Mo Haiwei B. amyloliquefaciens alkaline protease genes:AY665611.1) directly Synthesize target gene.Two primer (R are designed according to the target gene of synthesis：5'- ATCGGGATCCGCTCAACCGGCGAAAAATGTT-3' and F：5'-TCTAGCGGCCGC TTATTGAGCGGCAGCTTCGAC- 3') it is used to expand Mo Haiwei B. amyloliquefaciens alkaline Cathepsin B mP genes.The PCR product of amplification is purified into recycling, is connected to expression Carrier phyP43L, obtains expression vector phyP43L-BmP。
Embodiment 2, fixed point saturation mutation
193rd and the 288th influence to alkali protease BmP heat endurances are studied by saturation mutation.Fixed point is full It is as follows with the process of mutation：With the phyP built43L-BmP is template, and PCR amplification is carried out with corresponding mutant primer；It will expand The PCR product increased carries out agarose electrophoresis and purifies recycling PCR product.Original plasmid is divided with restriction enzyme DpnI Solution, Escherichia coli Top10 is transferred to by the product decomposed with heat shock method, and recombinant conversion, extraction verification are verified by bacterium solution PCR The plasmid of correct transformant is sequenced, so that it is determined that corresponding mutant.Correct mutant will be sequenced, converted by electricity It is transferred to bacillus subtilis WB600.
The screening of recombinant conversion is as follows：First by the recombinant bacterium grown on kalamycin resistance plate access LBK cultures In base, 37 DEG C, when 200rpm cultures 24 are small；By the bacterium solution centrifuging and taking supernatant after culture, enzyme activity determination, enzyme activity determination method are carried out Carried out with reference to national standard 2009；At 75 DEG C, water bath processing measures the heat endurance of mutant under the conditions of 5 minutes.Experimental result such as table 1 It is shown, as shown in Table 1：Two mutating acids are screened after 193 saturation mutations can improve the heat endurance of BmP, respectively It is S193L and S193K, retention rate is respectively 30% and 28%；288 also screen two mutating acids and can effectively improve The heat endurance of BmP, is S288M and S288K respectively, and retention rate is respectively 41% and 38%
1 original alkaline Cathepsin B mP of table and mutant heat endurance
Numbering Retention rate (%)
Original alkaline Cathepsin B mP 8
S193L 30
S193K 28
S288M 41
S288K 38
Embodiment 3, combinatorial mutagenesis
Mutation is combined on the basis of simple point mutation, finally obtaining 4 combinatorial mutagenesises by experiment is respectively designated as BmP1、BmP2、BmP3、BmP4.The mutational site that wherein BmP1 is included is S193K and S288M；The mutational site that BmP2 is included is S193L and S288M；The mutational site that BmP3 is included is S193L and S288K；The mutational site that BmP3 is included for S193K and S288K。
The thermal stability analysis of embodiment 4, original alkaline Cathepsin B mP and mutant
In order to accurately compare the heat endurance of original alkaline Cathepsin B mP and mutant, first to corresponding protease into Row purifying, purification process is ni-sepharose purification.By purified alkali protease BmP and mutant, at 75 DEG C, water bath processing 5 is divided Heat endurance is measured under the conditions of clock.By testing the guarantor of final definite mutant BmP1, BmP2, BmP3 and BmP4 under this condition It is respectively 65%, 60%, 56% and 49% to stay rate, and original alkaline Cathepsin B mP is 9%.
2 original alkaline Cathepsin B mP of table and combination mutant heat endurance
Numbering Retention rate (%)
Original alkaline Cathepsin B mP 9
BmP1：S193K/S288M 65
BmP2：S193L/S288M 60
BmP3：S193L/S288K 56
BmP3：S193K/S288K 49
Optimal reaction pH and the pH stability of embodiment 5, original alkaline Cathepsin B mP and mutant
With reference to the most suitable anti-of national standard method measure original alkaline Cathepsin B mP and mutant BmP1, BmP2, BmP3 and BmP4 Answer pH.The optimal reaction pH of original alkaline Cathepsin B mP and mutant BmP1, BmP2, BmP3 and BmP4 are as shown in Figure 1.By Fig. 1 Understand, the optimal pH of mutant BmP1, BmP2, BmP3 and BmP4 are almost 10.0 as original alkaline Cathepsin B mP.
By original alkaline Cathepsin B mP and mutant BmP1, BmP2, BmP3 and BmP4 room temperature under the conditions of pH6-11 respectively Handle 2 it is small when, referring next to state calibration method measure enzyme activity, the results are shown in Figure 2.As shown in Figure 2 mutant BmP1, BmP2, The pH stability of BmP3 and BmP4 is consistent with original alkaline Cathepsin B mP.
The optimal reactive temperature of embodiment 6, alkali protease BmP and mutant
With reference to the most suitable anti-of national standard method measure original alkaline Cathepsin B mP and mutant BmP1, BmP2, BmP3 and BmP4 Temperature is answered, the results are shown in Figure 3.From the figure 3, it may be seen that the optimal reactive temperature of alkali protease BmP is 65 DEG C, and mutant The optimal reactive temperature of BmP1, BmP2, BmP3 and BmP4 are 70 DEG C.
Sequence table
<110>Liu Danni
<120>Improve alkali protease BmP mutant and its gene and the application of heat endurance
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1140
<212> DNA
<213>Mo Haiwei bacillus (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>Mo Haiwei bacillus (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. improve heat endurance alkali protease BmP mutant, it is characterised in that the mutant be by amino acid sequence such as 193 of the mutant of alkali protease BmP shown in SEQ ID NO.6 sport L or K by amino acid S；Alternatively, 288 serve as reasons Amino acid S sports K or M and obtains.
2. encode the gene for the alkali protease BmP mutant that heat endurance is improved described in claim 1.
3. include the recombinant vector of gene described in claim 2.
4. include the host cell of gene described in claim 2.
A kind of 5. method for obtaining the alkali protease BmP mutant for improving heat endurance, it is characterised in that the described method includes By 193 of the mutant of alkali protease BmP of the amino acid sequence as shown in SEQ ID NO.6 by amino acid S sport L or K；Alternatively, 288 be the step of sporting K or M by amino acid S.
6. the application of the alkali protease BmP mutant of heat endurance is improved described in claim 1.
A kind of 7. method for preparing the alkali protease BmP mutant for improving heat endurance of fermenting, it is characterised in that the method Include the use of the step of recombinant vector described in claim 3 is fermented.
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CN108570461A (en) * 2018-04-17 2018-09-25 横琴仲泰生物医药有限公司 A kind of alkali protease BmP mutant and its encoding gene improving Rate activity
CN110923221A (en) * 2019-12-13 2020-03-27 中国科学院天津工业生物技术研究所 Novel alkaline protease high-temperature mutant from bacillus licheniformis
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
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

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US20050281773A1 (en) * 2002-12-20 2005-12-22 Henkel Kommanditgesellschaft Auf Aktien Subtilisin variants with improved perhydrolase activity
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