CN114561375A - Protease mutant BLAPR2 with improved heat stability and coding gene and application thereof - Google Patents

Protease mutant BLAPR2 with improved heat stability and coding gene and application thereof Download PDF

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CN114561375A
CN114561375A CN202210237403.2A CN202210237403A CN114561375A CN 114561375 A CN114561375 A CN 114561375A CN 202210237403 A CN202210237403 A CN 202210237403A CN 114561375 A CN114561375 A CN 114561375A
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CN114561375B (en
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肖志壮
方安然
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Qingdao Genyuan Biological Technology Group Co ltd
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Qingdao Red Cherry Biotechnology Co ltd
Qingdao Shangde Biotechnology 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Abstract

The invention provides a protease mutant BLAPR2 with improved thermal stability, and a coding gene and application thereof, and the invention uses an error-prone PCR method toBacillus licheniformisThe protease mutant BLAPR2 is obtained by mutating the protease gene and then screening the protease gene with high flux, compared with the unmutated protease, the thermal stability of the protease mutant obtained by the invention is obviously improved, and the protease mutant has good market application prospect and industrial value.

Description

Protease mutant BLAPR2 with improved heat stability as well as coding gene and application thereof
Technical Field
The invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a protease mutant BLAPR2 with improved thermal stability, and a coding gene and application thereof.
Background
Proteases are enzymes which catalyze the hydrolysis of peptide bonds in proteins, are widely present in animals, plants and microorganisms, have many different physiological functions, and are the first and most mature ones for the development of enzymology. The application of protease in various fields, such as food industry, brewing, detergent industry, feed industry, tanning industry, silk industry, pharmaceutical industry and the like, is more closely related to our life, and the environment puts higher demands on the yield and the characteristic improvement of protease.
At present, most of proteases on the market only have less than 20% of residual enzyme activity after being treated at 70 ℃, and the overall temperature resistance is poor, so that the wide use of the proteases is limited. For example, in the production of feed, the enzyme preparation is mixed with feed and then granulated at high temperature, during which the enzyme is easily inactivated. It is therefore of great importance to improve the thermostability of the protease.
The error-prone PCR technology is that when a target fragment is amplified by adopting DNA polymerase to carry out PCR reaction, the mutation frequency in the amplification process is increased by adjusting the reaction conditions, so that mutation is randomly introduced into a target gene at a certain frequency to construct a mutant library, and thus, a required forward mutant is screened. The error-prone PCR technique can be well applied to molecular modification of proteins.
Disclosure of Invention
The invention provides a protease mutant BLAPR2 with improved thermal stability, and a coding gene and application thereof, which are obtained by constructing a mutant library and performing directional screeningBacillus licheniformis And the protease gene from WX-02 is improved, and a mutant with improved heat stability is obtained by screening, so that the application effect of the mutant in the fields of feed, food or washing is improved.
In order to achieve the purpose of the invention, the invention is realized by adopting the following technical scheme:
the invention provides a protease mutant BLAPR2 with improved thermal stability, and the amino acid sequence of the protease mutant BLAPR2 is shown in SEQ ID NO: 5, the nucleotide sequence of the coding gene is shown as SEQ ID NO: and 6.
The invention also provides a recombinant expression vector containing the protease mutant coding gene.
The invention provides a genetic engineering bacterium containing a protease mutant coding gene, wherein the genetic engineering bacterium is bacillus subtilis and bacillus licheniformis.
The invention provides a preparation method of the protease mutant, which comprises the following steps:
1) constructing recombinant gene engineering bacteria: connecting the coding gene of the protease mutant to a pUB110 vector, transforming the recombinant vector into bacillus subtilis, and screening positive clones by using a resistance marker;
2) shaking flask fermentation of recombinant genetic engineering bacteria: inoculating the positive clone which is verified to be correct into a shake flask for fermentation, carrying out shake culture, and fermenting to generate a protease mutant;
3) amplifying and fermenting the recombinant strain: inoculating the gene engineering strain expressing the protease mutant into a fermentation tank, thereby producing the protease mutant BLAPR2 through fermentation.
Further: the fermentation medium in the step (3) comprises the following components: 5-10% of soybean meal, 1-5% of corn flour, 1-1.0% of PPG-200000.1, 0.1-1.0% of protease, 0.1-1.0% of amylase and 0.2-0.5% of disodium hydrogen phosphate by mass ratio.
The invention provides application of the protease mutant in preparation of feed additives, food additives and detergents.
Compared with the prior art, the invention has the advantages and the technical effects that: the invention is provided withBacillus licheniformis Based on the protease gene derived from WX-02, single-site mutant BLAPR1 containing A196C, double-site mutants BLAPR2 and BLAPR3 containing I140C/A196C and K49E/A196C respectively, and triple-site mutant BLAPR4 containing S191C/A196C/G308E are provided.
The improved mutant of the invention, namely the BLAPR1, BLAPR2, BLAPR3 and BLAPR4, has 30.2 percent, 64.0 percent, 45.9 percent and 39.0 percent of improved thermal stability when treated for 3 minutes at 75 ℃ compared with the original protease respectively. The thermal stability is obviously improved.
Therefore, the heat stability of the protease mutant obtained by the technical scheme of the invention is greatly improved compared with that of a wild type, so that the protease mutant has good application potential in the fields of feed, food or washing and the like. Has good market application prospect.
Drawings
FIG. 1 shows the fermentation data of the protease of the present invention in a 30L fermenter.
FIG. 2 shows the comparison of the heat resistance of the protease mutants of the present invention in a water bath at 75 ℃ for 3 min.
FIG. 3 shows the effect of the protease mutants of the present invention on the degradation of soybean meal resistant protein (1, 19 are protein markers; 2, 8, 12, 18 are soybean meal without enzyme addition; 3-7 are, respectively, 200U/g of BLAPR0, BLAPR1, BLAPR2, BLAPR3 and BLAPR 4; 9-13 are, respectively, 400U/g of BLAPR0, BLAPR1, BLAPR2, BLAPR3 and BLAPR 4; and 13-17 are, respectively, 600U/g of BLAPR0, BLAPR1, BLAPR2, BLAPR3 and BLAPR 4).
Detailed Description
In order to facilitate understanding of the invention, the invention will be described in more complete detail with reference to the drawings and examples, but the scope of the invention is not limited to the following examples.
The molecular biological experiments, which are not specifically described in the following examples, can be performed by referring to the specific methods listed in molecular cloning, A laboratory Manual (third edition) J. SammBruker, or according to the kit and product instructions. Reagents and biomaterials used in specific examples are commercially available without specific recitation.
1 strains and vectors
Bacillus subtilis WB600, plasmid pUB110, Escherichia coli BL21, plasmid pET-21a (+) were purchased from Invitrogen.
2 reagents and culture media
Plasmid extraction kit, fragment purification recovery kit, restriction enzyme and the like are purchased from precious bioengineering (Dalian) Co., Ltd; the GeneMorph II random mutation PCR kit was purchased from Stratagene; ampicillin, IPTG, etc. were purchased from Biotechnology engineering (Shanghai) Co., Ltd; protein Marker: blue Plus II Protein Marker (14-120 kDa) was purchased from GenBank technologies, Inc. of Beijing. LB culture medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.
Example 1: error-prone PCR construction of protease mutant library
Reference toBacillus licheniformis The amino acid sequence (SEQ ID NO: 1) and DNA sequence (SEQ ID NO: 2) of the WX-02-derived protease were designed as primers, Xba I restriction sites were designed at the 5 'end, and BamH I restriction sites were designed at the 3' end.
SEQ ID NO：1
MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
SEQ ID NO：2
ATGATGAGGAAGAAATCATTTTGGTTAGGGATGCTGACGGCGTTTATGTTAGTGTTTACG
ATGGCGTTTTCAGATAGCGCTTCTGCTGCACAACCTGCGAAAAATGTTGAAAAAGATTAT
ATCGTGGGGTTTAAATCTGGAGTTAAAACGGCGTCTGTGAAAAAAGATATTATTAAAGAA
TCAGGCGGCAAAGTCGATAAACAGTTTCGGATTATCAATGCTGCGAAAGCGAAACTTGAT
AAAGAAGCATTGAAAGAAGTCAAAAATGATCCGGATGTTGCTTACGTCGAAGAAGATCAT
GTCGCACATGCACTTGCTCAGACGGTGCCGTATGGCATCCCTCTTATCAAAGCAGATAAA
GTCCAAGCACAAGGCTTTAAAGGCGCTAATGTCAAAGTCGCGGTCCTTGATACGGGAATC
CAAGCAAGTCATCCGGATCTTAATGTGGTTGGGGGTGCGTCATTTGTCGCGGGAGAAGCA
TATAATACAGATGGCAACGGTCATGGAACACATGTTGCGGGAACGGTCGCAGCGTTAGAT
AATACGACGGGTGTGCTTGGTGTTGCACCGTCTGTCTCACTGTATGCGGTGAAAGTCCTT
AATTCTAGCGGATCTGGATCTTATTCAGGAATTGTGTCTGGAATCGAATGGGCTACAACG
AATGGCATGGATGTCATCAATATGAGCCTGGGAGGCGCGAGCGGCTCTACAGCTATGAAA
CAAGCAGTCGATAATGCGTATGCGCGCGGTGTTGTGGTGGTCGCAGCTGCGGGCAATTCA
GGCTCATCTGGCAATACGAATACGATCGGCTATCCGGCTAAATATGATTCAGTCATTGCT
GTGGGCGCGGTCGATTCTAATTCTAATCGTGCTTCTTTTAGCTCAGTGGGCGCAGAACTT
GAAGTGATGGCACCGGGCGCTGGAGTGTATAGCACCTATCCGACAAATACCTATGCTACA
CTGAATGGCACGTCTATGGCTTCACCTCATGTTGCAGGCGCCGCCGCTCTTATCCTGAGC
AAACATCCTAATTTGAGCGCGAGCCAGGTTCGTAATAGACTTTCTTCAACAGCGACGTAT
TTGGGCTCTAGCTTTTATTATGGCAAAGGACTGATCAATGTCGAAGCAGCTGCACAGTAA
Using GeneMorph II random mutation PCR kit, the PCR primers shown in SEQ ID NO: 2 as template, and carrying out random mutation by using the following primer sequences:
BLAPR-F：GCTCTAGAATGATGAGGAAGAAATC（SEQ ID NO：11）；
BLAPR-R：CGCGGATCCTTACTGTGCAGCTGCTTCGA（SEQ ID NO：12）。
and carrying out double enzyme digestion on the amplified random mutation PCR product by using Xba I and BamH I, purifying and recycling the product, connecting the product to a pET-21a (+) vector, transforming escherichia coli BL21-DE3, and screening positive clones by using an ampicillin-resistant LB plate to obtain pET-BLAPREX. The synthesized original gene was ligated to pET-21a (+) vector and transformed into E.coli BL21-DE3 to obtain pET-BLAPR0 in the same manner.
The selected single colonies were inoculated into a 96-well deep-well plate. Each plate was inoculated with 2 single colonies expressing BLAPR0 as controls. 300uL of LB liquid medium (containing 100. mu.g/mL ampicillin) was placed in each well, shake-cultured at 37 ℃ and 200rpm for 4 hours, 50uL of the bacterial solution was transferred to a new 96-well plate for stock preservation, 200uL of LB-Amp medium containing IPTG was added to the remaining bacterial solution on the plate so that the final concentration of IPTG was 1mM and the final concentration of ampicillin was 100. mu.g/mL, and shake-cultured at 37 ℃ and 200rpm for 10 hours to induce expression of protease.
And (3) repeatedly freezing and thawing the induced bacterial liquid for crushing, centrifuging the crushed cell sap, taking the supernatant, carrying out heat treatment (75 ℃ water bath for 3min), and then detecting the residual activity of the protease. And sequencing the mutant gene with the residual enzyme activity higher than that of the control.
The mutant A196C (named as BLAPR1) with improved heat resistance and taking the BLAPR0 as a starting template is screened, and the amino acid sequence is shown as SEQ ID NO: 3, and the coded nucleotide sequence is shown as SEQ ID NO: 4, respectively.
SEQ ID NO：3
MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYCVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
SEQ ID NO：4
ATGATGAGGAAGAAATCATTTTGGTTAGGGATGCTGACGGCGTTTATGTTAGTGTTTACG
ATGGCGTTTTCAGATAGCGCTTCTGCTGCACAACCTGCGAAAAATGTTGAAAAAGATTAT
ATCGTGGGGTTTAAATCTGGAGTTAAAACGGCGTCTGTGAAAAAAGATATTATTAAAGAA
TCAGGCGGCAAAGTCGATAAACAGTTTCGGATTATCAATGCTGCGAAAGCGAAACTTGAT
AAAGAAGCATTGAAAGAAGTCAAAAATGATCCGGATGTTGCTTACGTCGAAGAAGATCAT
GTCGCACATGCACTTGCTCAGACGGTGCCGTATGGCATCCCTCTTATCAAAGCAGATAAA
GTCCAAGCACAAGGCTTTAAAGGCGCTAATGTCAAAGTCGCGGTCCTTGATACGGGAATC
CAAGCAAGTCATCCGGATCTTAATGTGGTTGGGGGTGCGTCATTTGTCGCGGGAGAAGCA
TATAATACAGATGGCAACGGTCATGGAACACATGTTGCGGGAACGGTCGCAGCGTTAGAT
AATACGACGGGTGTGCTTGGTGTTGCACCGTCTGTCTCACTGTATTGCGTGAAAGTCCTT
AATTCTAGCGGATCTGGATCTTATTCAGGAATTGTGTCTGGAATCGAATGGGCTACAACG
AATGGCATGGATGTCATCAATATGAGCCTGGGAGGCGCGAGCGGCTCTACAGCTATGAAA
CAAGCAGTCGATAATGCGTATGCGCGCGGTGTTGTGGTGGTCGCAGCTGCGGGCAATTCA
GGCTCATCTGGCAATACGAATACGATCGGCTATCCGGCTAAATATGATTCAGTCATTGCT
GTGGGCGCGGTCGATTCTAATTCTAATCGTGCTTCTTTTAGCTCAGTGGGCGCAGAACTT
GAAGTGATGGCACCGGGCGCTGGAGTGTATAGCACCTATCCGACAAATACCTATGCTACA
CTGAATGGCACGTCTATGGCTTCACCTCATGTTGCAGGCGCCGCCGCTCTTATCCTGAGC
AAACATCCTAATTTGAGCGCGAGCCAGGTTCGTAATAGACTTTCTTCAACAGCGACGTAT
TTGGGCTCTAGCTTTTATTATGGCAAAGGACTGATCAATGTCGAAGCAGCTGCACAGTAA
Example 2: second round error-prone PCR construction of mutant library of protease BLAPR1
The protease gene BLAPR1 screened in the example 1 is used as a template, and the second round of random mutation, the construction process of a mutation library, the use of material reagents, the operation conditions and the like are the same as those in the example 1; and (3) performing mutant culture and screening by taking the BLAPR1 as a control, detecting the residual activity of the protease mutant after heat treatment (water bath at 75 ℃ for 3min), and sequencing the mutant gene with the residual activity higher than that of the BLAPR 1.
The following mutants with improved thermostability were finally screened:
the mutation mode of the BLAPR2 is I140C/A196C, and the amino acid sequence is shown as SEQ ID No: 5, the gene sequence is shown as SEQ ID No: 6 is shown in the specification;
the mutation mode of the BLAPR3 is K49E/A196C, and the amino acid sequence is shown as SEQ ID No: 7, and the gene sequence is shown as SEQ ID No: 8 is shown in the specification;
the mutation mode of the BLAPR4 is S191C/A196C/G308E, and the amino acid sequence is shown as SEQ ID No: 9, and the gene sequence is shown as SEQ ID No: shown at 10.
SEQ ID No：5
MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGCQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYCVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
SEQ ID No：6
ATGATGAGGAAGAAATCATTTTGGTTAGGGATGCTGACGGCGTTTATGTTAGTGTTTACG
ATGGCGTTTTCAGATAGCGCTTCTGCTGCACAACCTGCGAAAAATGTTGAAAAAGATTAT
ATCGTGGGGTTTAAATCTGGAGTTAAAACGGCGTCTGTGAAAAAAGATATTATTAAAGAA
TCAGGCGGCAAAGTCGATAAACAGTTTCGGATTATCAATGCTGCGAAAGCGAAACTTGAT
AAAGAAGCATTGAAAGAAGTCAAAAATGATCCGGATGTTGCTTACGTCGAAGAAGATCAT
GTCGCACATGCACTTGCTCAGACGGTGCCGTATGGCATCCCTCTTATCAAAGCAGATAAA
GTCCAAGCACAAGGCTTTAAAGGCGCTAATGTCAAAGTCGCGGTCCTTGATACGGGATGC
CAAGCAAGTCATCCGGATCTTAATGTGGTTGGGGGTGCGTCATTTGTCGCGGGAGAAGCA
TATAATACAGATGGCAACGGTCATGGAACACATGTTGCGGGAACGGTCGCAGCGTTAGAT
AATACGACGGGTGTGCTTGGTGTTGCACCGTCTGTCTCACTGTATTGCGTGAAAGTCCTT
AATTCTAGCGGATCTGGATCTTATTCAGGAATTGTGTCTGGAATCGAATGGGCTACAACG
AATGGCATGGATGTCATCAATATGAGCCTGGGAGGCGCGAGCGGCTCTACAGCTATGAAA
CAAGCAGTCGATAATGCGTATGCGCGCGGTGTTGTGGTGGTCGCAGCTGCGGGCAATTCA
GGCTCATCTGGCAATACGAATACGATCGGCTATCCGGCTAAATATGATTCAGTCATTGCT
GTGGGCGCGGTCGATTCTAATTCTAATCGTGCTTCTTTTAGCTCAGTGGGCGCAGAACTT
GAAGTGATGGCACCGGGCGCTGGAGTGTATAGCACCTATCCGACAAATACCTATGCTACA
CTGAATGGCACGTCTATGGCTTCACCTCATGTTGCAGGCGCCGCCGCTCTTATCCTGAGC
AAACATCCTAATTTGAGCGCGAGCCAGGTTCGTAATAGACTTTCTTCAACAGCGACGTAT
TTGGGCTCTAGCTTTTATTATGGCAAAGGACTGATCAATGTCGAAGCAGCTGCACAGTAA
SEQ ID No：7
MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVETASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYCVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
SEQ ID No：8
ATGATGAGGAAGAAATCATTTTGGTTAGGGATGCTGACGGCGTTTATGTTAGTGTTTACG
ATGGCGTTTTCAGATAGCGCTTCTGCTGCACAACCTGCGAAAAATGTTGAAAAAGATTAT
ATCGTGGGGTTTAAATCTGGAGTTGAAACGGCGTCTGTGAAAAAAGATATTATTAAAGAA
TCAGGCGGCAAAGTCGATAAACAGTTTCGGATTATCAATGCTGCGAAAGCGAAACTTGAT
AAAGAAGCATTGAAAGAAGTCAAAAATGATCCGGATGTTGCTTACGTCGAAGAAGATCAT
GTCGCACATGCACTTGCTCAGACGGTGCCGTATGGCATCCCTCTTATCAAAGCAGATAAA
GTCCAAGCACAAGGCTTTAAAGGCGCTAATGTCAAAGTCGCGGTCCTTGATACGGGAATC
CAAGCAAGTCATCCGGATCTTAATGTGGTTGGGGGTGCGTCATTTGTCGCGGGAGAAGCA
TATAATACAGATGGCAACGGTCATGGAACACATGTTGCGGGAACGGTCGCAGCGTTAGAT
AATACGACGGGTGTGCTTGGTGTTGCACCGTCTGTCTCACTGTATTGCGTGAAAGTCCTT
AATTCTAGCGGATCTGGATCTTATTCAGGAATTGTGTCTGGAATCGAATGGGCTACAACG
AATGGCATGGATGTCATCAATATGAGCCTGGGAGGCGCGAGCGGCTCTACAGCTATGAAA
CAAGCAGTCGATAATGCGTATGCGCGCGGTGTTGTGGTGGTCGCAGCTGCGGGCAATTCA
GGCTCATCTGGCAATACGAATACGATCGGCTATCCGGCTAAATATGATTCAGTCATTGCT
GTGGGCGCGGTCGATTCTAATTCTAATCGTGCTTCTTTTAGCTCAGTGGGCGCAGAACTT
GAAGTGATGGCACCGGGCGCTGGAGTGTATAGCACCTATCCGACAAATACCTATGCTACA
CTGAATGGCACGTCTATGGCTTCACCTCATGTTGCAGGCGCCGCCGCTCTTATCCTGAGC
AAACATCCTAATTTGAGCGCGAGCCAGGTTCGTAATAGACTTTCTTCAACAGCGACGTAT
TTGGGCTCTAGCTTTTATTATGGCAAAGGACTGATCAATGTCGAAGCAGCTGCACAGTAA
SEQ ID No：9
MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPCVSLYCVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAEVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEAAAQ
SEQ ID No：10
ATGATGAGGAAGAAATCATTTTGGTTAGGGATGCTGACGGCGTTTATGTTAGTGTTTACG
ATGGCGTTTTCAGATAGCGCTTCTGCTGCACAACCTGCGAAAAATGTTGAAAAAGATTAT
ATCGTGGGGTTTAAATCTGGAGTTAAAACGGCGTCTGTGAAAAAAGATATTATTAAAGAA
TCAGGCGGCAAAGTCGATAAACAGTTTCGGATTATCAATGCTGCGAAAGCGAAACTTGAT
AAAGAAGCATTGAAAGAAGTCAAAAATGATCCGGATGTTGCTTACGTCGAAGAAGATCAT
GTCGCACATGCACTTGCTCAGACGGTGCCGTATGGCATCCCTCTTATCAAAGCAGATAAA
GTCCAAGCACAAGGCTTTAAAGGCGCTAATGTCAAAGTCGCGGTCCTTGATACGGGAATC
CAAGCAAGTCATCCGGATCTTAATGTGGTTGGGGGTGCGTCATTTGTCGCGGGAGAAGCA
TATAATACAGATGGCAACGGTCATGGAACACATGTTGCGGGAACGGTCGCAGCGTTAGAT
AATACGACGGGTGTGCTTGGTGTTGCACCGTGCGTCTCACTGTATTGCGTGAAAGTCCTT
AATTCTAGCGGATCTGGATCTTATTCAGGAATTGTGTCTGGAATCGAATGGGCTACAACG
AATGGCATGGATGTCATCAATATGAGCCTGGGAGGCGCGAGCGGCTCTACAGCTATGAAA
CAAGCAGTCGATAATGCGTATGCGCGCGGTGTTGTGGTGGTCGCAGCTGCGGGCAATTCA
GGCTCATCTGGCAATACGAATACGATCGGCTATCCGGCTAAATATGATTCAGTCATTGCT
GTGGGCGCGGTCGATTCTAATTCTAATCGTGCTTCTTTTAGCTCAGTGGGCGCAGAACTT
GAAGTGATGGCACCGGGCGCTGAAGTGTATAGCACCTATCCGACAAATACCTATGCTACA
CTGAATGGCACGTCTATGGCTTCACCTCATGTTGCAGGCGCCGCCGCTCTTATCCTGAGC
AAACATCCTAATTTGAGCGCGAGCCAGGTTCGTAATAGACTTTCTTCAACAGCGACGTAT
TTGGGCTCTAGCTTTTATTATGGCAAAGGACTGATCAATGTCGAAGCAGCTGCACAGTAA
Example 3: expression verification of protease mutant with improved thermal stability in bacillus subtilis
The excellent mutants are respectively cloned into Xba I and BamH I sites of a plasmid pUB110, and the recombinant plasmid is transformed into Bacillus subtilis WB600 by referring to a Bacillus subtilis transformation formula created by Spizizen to obtain recombinant bacteria. According to a fermentation medium: 50-80g/L of soybean meal, 60-100g/L of corn starch, 2-4g/L of disodium hydrogen phosphate and 1-2g/L of sodium carbonate, wherein the pH is natural, after shaking fermentation is carried out for 78 hours, a culture solution is centrifuged to obtain a supernatant, the average enzyme activity of the supernatant of each mutant fermentation liquid is measured, a transformant with the highest enzyme activity in each mutant is taken for fermentation, and after the transformant is treated in a water bath at 75 ℃ for 3 minutes, the enzyme activity retention rate is compared, and the result is shown in figure 2. The thermal stability of the mutated proteases BLAPR1, BLAPR2, BLAPR3 and BLAPR4 after treatment at 75 ℃ for 3 minutes is improved by 30.2%, 64.0%, 45.9% and 39.0% respectively compared with the original protease.
The results show that the mutation of Ala at position 196 of BLAPR0 to Cys can improve the heat resistance of the enzyme while keeping the original enzyme activity. On the basis, mutation of Ile at position 140 into Cys or mutation of Lys at position 49 into Glu; or a mutant obtained by mutating Ser at the 191 th position to Cys and simultaneously mutating Gly at the 308 th position to Glu, and the heat resistance is further improved.
Example 4: protease mutants were fermented and prepared in a 30L fermentor
Genetically engineered bacteria expressing the protease mutants BLAPR0, BLAPR1, BLAPR2, BLAPR3 and BLAPR4 in the above examples were streaked on LB plates containing kanamycin resistance (20. mu.g/mL final concentration), cultured at 37 ℃ until single colonies grew out, and the single colonies growing well were selected and streaked on LB plates containing kanamycin resistance (20. mu.g/mL final concentration), and the colonies of recombinant Bacillus subtilis thus activated three generations were inoculated in 50mL of LB medium containing kanamycin resistance (20. mu.g/mL final concentration), and cultured at 37 ℃ and 200rpm for 24 hours. 2% of the inoculum size was inoculated into 1L LB medium containing 20. mu.g/mL kanamycin to a final concentration, 37 ℃, 200rpm culture to OD600 of about 5, used as seed liquid inoculation fermentation tank.
The fermentation production process comprises the following steps: 5-10% of soybean meal, 1-5% of corn flour, 1-1.0% of PPG-200000.1, 0.1-1.0% of protease, 0.1-1.0% of amylase, 0.2-0.5% of disodium hydrogen phosphate (12 water), natural pH, 37 ℃, stirring speed of 600rpm, ventilation volume of 1.5 (v/v), and dissolved oxygen controlled at more than 20%. The pH value is natural in the fermentation process, the enzyme activity is measured after fermentation is carried out for 24 hours, and after the fermentation is finished (generally 48 hours), the fermentation liquor is processed by a plate-and-frame filter to obtain crude enzyme liquid, and the crude enzyme liquid is sprayed and dried by a spray tower to form a powder preparation for application test.
Example 5: degradation experiment of protease mutant on resistance protein
(1) Resistant proteolysis reaction of soybean meal
Soybean meal substrate: taking 2g of crushed soybean meal sieved by a 60-mesh sieve, and adding 20mL of buffer solution (pH 8.00.02M Tris-HCl buffer solution) to prepare suspension;
experimental groups: and (3) centrifuging fermentation liquor of protease mutant BLAPR0, BLAPR1, BLAPR2, BLAPR3 and BLAPR4 genetically engineered bacteria to obtain fermentation liquor supernatant. Accurately measuring enzyme activity, and adding according to the same enzyme activity (200U/g, 400U/g and 600U/g);
control group: no enzyme is added, and the treatment process is consistent with that of the test group;
reaction conditions are as follows: uniformly mixing the enzyme solution and the substrate, and carrying out enzymolysis reaction for 2 hours at 37 ℃ and 120rpm/min on a water bath shaker;
after the reaction is finished, 0.5mL of the soybean meal enzymolysis liquid is added with 4.5mL of ELISA extracting solution, and the mixture is extracted for 16h at 25 ℃ and 200 rpm/min.
(2) SDS-PAGE examination of antigen protein removal
And (2) taking 100 mu L of the sample subjected to reaction and extraction in the step (1), adding 100 mu L of Loading Buffer, boiling for 10min, and taking 20 mu L of the sample for Loading.
Electrophoresis conditions: the voltage is 80-90mv and the current is about 30mA when the gel is concentrated; when separating the gel, the voltage is 110-120mv, and the current is about 40 mA. The sample is stopped at a position 1cm away from the edge of the gel. Stripping the glue, dyeing for 2h, and decolorizing for 24h, wherein the decolorizing solution is replaced.
The test results are shown in fig. 3: under the same conditions and with the same addition amount of enzyme activity units, the degradation effects of the protease mutants such as BLAPR0, BLAPR1, BLAPR2, BLAPR3 and BLAPR4 on the soybean meal are basically consistent; when the addition amount of the protease mutant is 400U/g, the effect of degrading the soybean meal is remarkable, and only a shallow belt with about 50KD (beta subunit) is left; when the addition amount of the protease mutant is 600U/g, the soybean meal can be completely degraded.
The experiment shows that the protease mutants of BLAPR1, BLAPR2, BLAPR3 and BLAPR4 maintain the original property of efficiently degrading resistant protein, and meanwhile, the heat stability of the protease mutants is obviously improved, so that the protease mutants of BLAPR1, BLAPR2, BLAPR3 and BLAPR4 have good application potential.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> Qingdao Shangde Biotechnology Co., Ltd
QINGDAO RED CHERRY BIOTECHNOLOGY Co.,Ltd.
<120> protease mutant BLAPR2 with improved heat stability, and coding gene and application thereof
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 379
<212> PRT
<213> Bacillus licheniformis (Bacillus licheniformis)
<400> 1
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly 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> 2
<211> 1140
<212> DNA
<213> Bacillus licheniformis (Bacillus licheniformis)
<400> 2
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacgacgg gtgtgcttgg tgttgcaccg tctgtctcac tgtatgcggt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 3
<211> 379
<212> PRT
<213> Bacillus licheniformis (Bacillus licheniformis)
<400> 3
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Cys 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> 4
<211> 1140
<212> DNA
<213> Bacillus licheniformis (Bacillus licheniformis)
<400> 4
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacgacgg gtgtgcttgg tgttgcaccg tctgtctcac tgtattgcgt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 5
<211> 379
<212> PRT
<213> Bacillus licheniformis (Bacillus licheniformis)
<400> 5
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Cys 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 Cys 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> 6
<211> 1140
<212> DNA
<213> Bacillus licheniformis (Bacillus licheniformis)
<400> 6
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggatgc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacgacgg gtgtgcttgg tgttgcaccg tctgtctcac tgtattgcgt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 7
<211> 379
<212> PRT
<213> Bacillus licheniformis (Bacillus licheniformis)
<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 Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Glu 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 Cys 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> 8
<211> 1140
<212> DNA
<213> Bacillus licheniformis (Bacillus licheniformis)
<400> 8
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttgaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacgacgg gtgtgcttgg tgttgcaccg tctgtctcac tgtattgcgt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 9
<211> 379
<212> PRT
<213> Bacillus licheniformis (Bacillus licheniformis)
<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 Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Cys Val
180 185 190
Ser Leu Tyr Cys 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 Glu 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> 1140
<212> DNA
<213> Bacillus licheniformis (Bacillus licheniformis)
<400> 10
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacgacgg gtgtgcttgg tgttgcaccg tgcgtctcac tgtattgcgt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tgaagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 11
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gctctagaat gatgaggaag aaatc 25
<210> 12
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cgcggatcct tactgtgcag ctgcttcga 29

Claims (7)

1. A protease mutant BLAPR2 with improved thermostability, having an amino acid sequence as set forth in SEQ ID NO: 5, the nucleotide sequence of the coding gene is shown as SEQ ID NO: and 6.
2. A recombinant expression vector comprising a gene encoding the protease mutant of claim 1.
3. A genetically engineered bacterium comprising a gene encoding the protease mutant of claim 1, wherein the genetically engineered bacterium is Bacillus subtilis or Bacillus licheniformis.
4. The method for producing the protease mutant according to claim 1, comprising the steps of:
1) constructing recombinant gene engineering bacteria: connecting the coding gene of the protease mutant to a pUB110 vector, transforming the recombinant vector into bacillus subtilis, and screening positive clones by using a resistance marker;
2) shaking flask fermentation of recombinant gene engineering bacteria: inoculating the positive clone which is verified to be correct into a shake flask for fermentation, carrying out shake culture, and fermenting to generate a protease mutant;
3) amplifying and fermenting the recombinant strain: inoculating the gene engineering strain expressing the protease mutant into a fermentation tank, thereby producing the protease mutant BLAPR2 through fermentation.
5. The method for preparing the protease mutant according to claim 4, wherein the fermentation medium of step (3) comprises the following components: 5-10% of soybean meal, 1-5% of corn flour, 1-1.0% of PPG-200000.1, 0.1-1.0% of protease, 0.1-1.0% of amylase and 0.2-0.5% of disodium hydrogen phosphate by mass ratio.
6. Use of the protease mutant according to claim 1 for the preparation of feed additives and food additives.
7. Use of the protease mutant according to claim 1 for the preparation of detergents.
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