CN108384771A - A kind of alkali protease mutation body and its encoding gene improving Rate activity - Google Patents

A kind of alkali protease mutation body and its encoding gene improving Rate activity Download PDF

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Publication number
CN108384771A
CN108384771A CN201810119791.8A CN201810119791A CN108384771A CN 108384771 A CN108384771 A CN 108384771A CN 201810119791 A CN201810119791 A CN 201810119791A CN 108384771 A CN108384771 A CN 108384771A
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CN108384771B (en
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刘丹妮
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Zhuhai Double Ring Investment Co Ltd
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Zhuhai Double Ring Investment Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 genetic engineering fields, and in particular to a kind of alkali protease BmP mutant and its encoding gene improving Rate activity.The mutational site of alkali protease BmP mutant of the present invention is that the 178th of alkali protease BmP shown in amino acid SEQ ID NO.7 is mutated into D or M or G or Q or T by S.The mutant that the present invention obtains has higher than living than protoenzyme.

Description

A kind of alkali protease mutation body and its encoding gene improving Rate activity
Technical field
The present invention relates to protein molecules, and field is transformed, and in particular to a kind of alkali protease BmP improving Rate activity is prominent Variant and its encoding gene and application.
Background technology
Protease is the class of enzymes of catalytic proteins hydrolysis, can protease be divided into acid according to its optimal reaction pH environment Property protease, neutral proteinase and alkali protease.Alkali protease is the enzyme of a kind of aminosal under alkaline condition Class, alkali protease are widely used in the industrial circles such as food, washing, feed because having stronger hydrolysis ability.At present Alkali protease is mainly derived from bacillus licheniformis, bacillus subtilis and Bacillus circulans, other bacillus alkali Property protease reporter is relatively fewer.
In the experiment of early period, this laboratory clone is expressed and is analyzed from Mo Haiwei bacillus The alkali protease BmP, BmP of (Bacillus mojavensis) are strong to protein raw materials capacity of decomposition, suitable for food, wash It washs, the industrial circles such as feed.But BmP Rate activities are relatively low, production cost is high, limits its commercial application.Therefore it carries The Rate activity of high BmP is BmP commercial application urgent problems.
Invention content
The present invention carries out egg to deriving from Mo Haiwei bacillus (Bacillus mojavensis) alkali protease BmP White matter molecular modification reduces its production cost to improve the Rate activity of BmP, lays the foundation for the industrial applications of BmP.
The nucleotide sequence and amino acid sequence of the alkali protease BmP of Mo Haiwei bacillus is respectively such as SEQ ID Shown in NO.1 and SEQ ID NO.7.
The object of the present invention is to provide a kind of mutant of raising Rate activity alkali protease BmP.The mutant is ammonia The mutant of base acid sequence Cathepsin B mP as shown in SEQ ID NO.7, wherein mutational site are the 178th.
The present invention obtains the tomograph of alkali protease BmP by homologous modeling first, and applicant is sent out by testing Existing, the hydrolysing activity potential contribution of the 178th couple of alkali protease BmP is larger, therefore selected 178th prominent as fixed point saturation Become target site.Using fixed point saturation mutation method to the 178th of alkali protease BmP shown in SEQ ID NO.7 into Row mutation, the Rate activity that 5 mutant can effectively improve alkali protease BmP is obtained by screening.This 5 mutant point B1, B2, B3, B4 and B5 are not named as it.Rate activity relative to original alkaline Cathepsin B mP, B1, B2, B3, B4 and B5 is distinguished Improve 35%, 21%, 28%, 19% and 25%.The nucleotide sequence of mutant B1, B2, B3, B4 and B5 such as SEQ ID Shown in NO.2 to SEQ ID NO.6, amino acid sequence is as shown in SEQ ID NO.8 to SEQ ID NO.12.Wherein B1 includes Mutational site is S178D；The mutational site that B2 includes is S178M；The mutational site that B3 includes is S178G；The mutation that B4 includes Site is S178Q；The mutational site that B5 includes is S178T.
According to having reported Mo Haiwei B. amyloliquefaciens alkaline proteinase gene sequences (Genebank:AY665611.1) directly Target gene is synthesized, Mo Haiwei B. amyloliquefaciens alkaline Cathepsin B mP genes are expanded.The PCR product purifications of amplification are recycled, even It is connected to expression vector phyP43L obtains expression vector phyP43L-BmP；By saturation mutation alkali protease BmP the 178th Obtain the mutant for improving Rate activity alkali protease BmP.
Wherein fixed point saturation mutation specifically comprises the following steps：With the phyP built43L-BmP is template, with corresponding Mutant primer carries out PCR amplification；The PCR product expanded is subjected to agarose electrophoresis and purifies recycling PCR product.With limitation Property restriction endonuclease DpnI original plasmid is decomposed, the product decomposed just with heat shock method is transferred to Escherichia coli Top10, passes through bacterium Liquid PCR verification recombinant conversion, the plasmid that correct transformant is verified in extraction is sequenced, so that it is determined that corresponding mutant. Correct mutant will be sequenced, withered bacillus subtilis WB600 is transferred to by electrotransformation.
Another object of the present invention is to provide the encoding gene for improving Rate activity alkali protease BmP mutant.The base The nucleotides sequence of cause is classified as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6 It is shown.
It includes the raising Rate activity alkali protease BmP mutant code genes that the present invention, which is also claimed a kind of, Recombinant vector.
The present invention is also claimed a kind of comprising the host for improving Rate activity alkali protease BmP mutant code genes Cell.
A kind of application improved than alkali protease BmP mutant living is also claimed in the present invention.
The present invention improves Mo Haiwei bacillus (Bacillus mojavensis) alkalinity by albumen design and rational The Rate activity of Cathepsin B mP reduces its production cost, lays the foundation for its industrial applications.
Description of the drawings
The optimal reaction pH of Fig. 1 original alkaline Cathepsin B mP and mutant B1 to B5.
The pH stability of Fig. 2 original alkaline Cathepsin B mP and mutant B1 to B5.
The optimal reactive temperature of Fig. 3 original alkaline Cathepsin B mP and mutant B1 to B5.
The thermal stability of Fig. 4 original alkaline Cathepsin B mP and mutant B1 to B5.
Specific implementation mode
Do not make the experimental methods of molecular biology illustrated, equal reference in following embodiment《Molecular Cloning: A Laboratory refers to South》Listed specific method carries out in one book of (third edition) J. Pehanorm Brookers, or according to kit and product description into Row；The reagent and biomaterial commercially obtain unless otherwise specified.Experiment material and reagent：
1, bacterial strain and carrier
Coli strain Topl0, bacillus subtilis WB600, expression vector phyP43L is by this Laboratories Accession.
2, 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 Shanghai Sangon Biotech Company.
3, culture medium
Escherichia coli culture medium is LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).Withered grass gemma Culture medium is that LBK is that LB culture mediums add kanamycins.
The clone of embodiment 1, Mo Haiwei bacillus (Bacillus mojavensis) alkali protease BmP genes
According to having reported Mo Haiwei B. amyloliquefaciens alkaline proteinase gene sequences (Genebank:AY665611.1) directly Synthesize target gene.Two primer (R are designed according to the target gene of synthesis：5'-ATCGGGATCC GCTCAACCGGCGAAAAATGTT-3' and F：5'-TCTAGCGGCCGC TTATTGAGCG GCAGCTTCGAC-3') for expanding Increase Mo Haiwei B. amyloliquefaciens alkaline Cathepsin B mP genes.The PCR product of amplification is purified into recycling, is connected to expression vector phyP43L obtains expression vector phyP43L-BmP。
Embodiment 2, design and rational pinpoint saturation mutation
Alkali protease BmP is modeled by homology modeling software, obtains the three-dimensional conformation figure of BmP.Pass through Bioinformatics software carries out docking analysis to the BmP models after structure, finds the 178th, key amino acid site.By full Influence with mutation research the 178th to alkali protease BmP Rate activities.
The process for pinpointing saturation mutation is as follows：With the phyP built43L-BmP is template, with corresponding mutant primer into Row PCR amplification；The PCR product expanded is subjected to agarose electrophoresis and purifies recycling PCR products.Use restriction enzyme DpnI decomposes original plasmid, and the product decomposed is just transferred to Escherichia coli Top10 with heat shock method, is verified by bacterium solution PCR Recombinant conversion, the plasmid that correct transformant is verified in extraction is sequenced, so that it is determined that corresponding mutant.It will be sequenced just True mutant is transferred to withered bacillus subtilis WB600 by electrotransformation.
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, 200rpm is cultivated 24 hours；By the bacterium solution centrifuging and taking supernatant after culture, enzyme activity determination, enzyme activity determination side are carried out Method is carried out with reference to GBT/28715-2012.
Embodiment 3, original alkaline Cathepsin B mP and the analysis of mutant Rate activity
Alkali protease BmP and mutant are purified using ni-sepharose purification.By alkali protease BmP after purification Rate activity measurement is carried out with mutant, experimental result is as shown in table 1, and the 178th is to influence alkali protease BmP as shown in Table 1 The key amino acid site of hydrolysing activity, when it sports D, M, G, Q and T, opposite Rate activity has been respectively increased 35%, 21%, 28%, 19% and 25%.
1 original alkaline Cathepsin B mP of table and the analysis of mutant Rate activity
Number Opposite Rate activity (%)
Original alkaline Cathepsin B mP 100
S178D 135
S178M 121
S178G 128
S178Q 119
S178T 125
Optimal reaction pH and the pH stability of embodiment 4, original alkaline Cathepsin B mP and mutant
The optimal reaction pH of original alkaline Cathepsin B mP and mutant B1 to B5, original alkaline are measured with reference to national standard method The optimal reaction pH of Cathepsin B mP and mutant B1 to B5 is as shown in Figure 2.As shown in Figure 2, the optimal pH of mutant B1 to B5 Value is almost 10.0 as original alkaline Cathepsin B mP.
By original alkaline Cathepsin B mP and mutant B1 to B5 respectively under the conditions of pH6-11 room temperature handle 2 hours, so Enzyme activity is measured with reference to state's calibration method afterwards, the results are shown in Figure 3.As shown in Figure 3 the pH stability of mutant B1 to B5 with it is original Alkali protease BmP is consistent.
The optimal reactive temperature and thermal stability of embodiment 6, alkali protease BmP and mutant
The optimal reactive temperature of original alkaline Cathepsin B mP and mutant B1 to B5 is measured with reference to national standard method, as a result such as Shown in Fig. 4.As shown in Figure 4, the optimal reactive temperature of alkali protease BmP is 65 DEG C, and the optimal reaction of mutant B1 to B5 Temperature is 60 DEG C.
By original alkaline Cathepsin B mP and mutant B1 to B5 respectively at 70 DEG C, water bath processing after five minutes, with reference to national standard Measure remaining enzyme activity.As shown in Figure 4, relative to alkali protease BmP (retention rate 40%), the thermostabilization of mutant B1 to B5 Property is declined, and remaining enzyme activity retention rate is respectively 31%, 32%, 36%, 33% and 39%.
Sequence table
<110>Two fingers ring Investment Co., Ltd of Zhuhai City
<120>A kind of alkali protease mutation body and its encoding gene improving Rate activity
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 1
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 atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg atcatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg 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 taa 1053
<210> 2
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 2
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 atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg agattacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg 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 taa 1053
<210> 3
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 3
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 atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg aatgtacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg 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 taa 1053
<210> 4
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 4
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 atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg aggttacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg 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 taa 1053
<210> 5
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<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 atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg acaatacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg 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 taa 1053
<210> 6
<211> 1053
<212> DNA
<213> Bacillus mojavensis
<400> 6
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 atccaagctt ctcatccgga cttgaacgta 360
gtcggcggag caagctttgt ggctggcgaa gcttataaca ccgacggcaa cggacacggc 420
acgcatgttg ccggtacagt agctgcgctt gacaatacaa cgggtgtatt aggcgttgcg 480
ccaagcgtat ccttgtacgc ggttaaagta ctgaattcaa gcggaagcgg aacctacagc 540
ggcattgtaa gcggaatcga gtgggcgaca acaaacggca tggatgttat caatatgagc 600
cttgggggag catcaggctc gacagcgatg aaacaggcag tcgacaatgc atatgcaaga 660
ggggttgtcg ttgtagctgc agcagggaac agcggatctt caggaaacac gaatacaatt 720
ggctatcctg cgaaatacga ttctgtcatc gctgttggtg 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 taa 1053
<210> 7
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 7
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
340 345 350
<210> 8
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 8
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 Asp 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
340 345 350
<210> 9
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 9
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 Met 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
340 345 350
<210> 10
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 10
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 Gly 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
340 345 350
<210> 11
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 11
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 Gln 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
340 345 350
<210> 12
<211> 350
<212> PRT
<213> Bacillus mojavensis
<400> 12
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 Thr 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
340 345 350

Claims (6)

1. it is a kind of raising than alkali protease BmP mutant living, which is characterized in that the mutant be amino acid sequence such as The mutant of Cathepsin B mP shown in SEQ ID NO.7, wherein mutational site are the 178th.
2. raising according to claim 1 is than alkali protease BmP mutant living, which is characterized in that amino acid sequence The mutational site of alkali protease BmP mutant as shown in SEQ ID NO.7 is the 178th and is mutated into D or M or G or Q by S Or T, amino acid sequence such as SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 or SEQ ID Shown in NO.12.
3. the gene than alkali protease BmP mutant living is improved described in a kind of coding claim 2, wherein the gene Nucleotides sequence be classified as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, shown in SEQ ID NO.5 or SEQ ID NO.6.
4. a kind of recombinant vector including gene described in claim 3.
5. a kind of host cell including gene described in claim 3.
6. improving the application than alkali protease BmP mutant living described in a kind of claim 2.
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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
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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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
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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