CN114196656B - Proteinase K mutant and construction and application of its expression vector - Google Patents

Proteinase K mutant and construction and application of its expression vector Download PDF

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CN114196656B
CN114196656B CN202111640121.9A CN202111640121A CN114196656B CN 114196656 B CN114196656 B CN 114196656B CN 202111640121 A CN202111640121 A CN 202111640121A CN 114196656 B CN114196656 B CN 114196656B
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proteinase
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CN114196656A (en
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刁含文
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Nanjing Jujiang Biotechnology Co ltd
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    • 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)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/102—Plasmid DNA for yeast

Abstract

The invention discloses a proteinase K mutant and construction and application of an expression vector thereof, relates to the technical field of bioengineering, and provides the proteinase K mutant which has at least one mutation as follows on the basis of an amino acid sequence shown in SEQ ID No. 1: F371C and T373P have better enzyme activity compared with wild type proteinase K, offer the way for application and study of proteinase K. In addition, the invention also provides the construction of the proteinase K mutant high-copy expression vector, improves the screening power of high-expression thalli, reduces the operation of a large number of screening bacteria, and indicates the direction for the industrial production route of proteinase K with application value.

Description

Proteinase K mutant and construction and application of its expression vector
Technical Field
The invention relates to the technical field of bioengineering, in particular to a proteinase K mutant and construction and application of an expression vector thereof.
Background
Proteinase K belongs to the class of serine proteases, which is a major class of proteases produced by Candida albicans (Tritirachium album Limber) at Lin Bashi. Since a microorganism capable of synthesizing the protease can grow in an environment using keratin (Kerantin) as a sole carbon and nitrogen source, it is called proteinase K, and its three-dimensional structure is shown in FIG. 1. Proteinase K has extremely high enzyme activity and wide substrate specificity, and can decompose ester bond and peptide bond adjacent to hydrophobic amino acid, sulfur-containing amino acid and aromatic amino acid at C terminal, and is used in degrading protein to produce short peptide. It has the characteristics of the typical catalytic triad Asp39-His69-Ser224 possessed by serine proteases and two Ca's around the active center 2+ Binding sites to increase their stability, see figure 2, allow them to maintain higher enzyme activity under a wider range of conditions. Proteinase K can inactivate or degrade proteins, using thisThe characteristic of the protease K has important application in the fields of nucleic acid purification, silk, medicine, food, brewing and the like.
Since Lin Bashi is slow to produce, there is a great limit to the practical production of proteinase K. Related researchers have attempted to express proteinase K in E.coli, but most of them exist in the form of inclusion bodies, which is disadvantageous for industrial production.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a proteinase K mutant and construction and application of an expression vector thereof.
The invention is realized in the following way:
in a first aspect, embodiments of the present invention provide a proteinase K mutant having at least one of the following mutations based on the amino acid sequence shown in SEQ ID No. 1: F371C and T373P.
In a second aspect, embodiments of the invention provide an isolated nucleic acid comprising a nucleic acid sequence encoding a proteinase K mutant as described in the previous embodiments.
In a third aspect, embodiments of the present invention provide a recombinant vector comprising an isolated nucleic acid as described in the previous embodiments.
In a fourth aspect, an embodiment of the present invention provides a method for constructing a recombinant vector according to the previous embodiment, which includes: inserting the separated nucleic acid into a vector, and constructing to obtain a single copy recombinant vector.
In a fifth aspect, an embodiment of the present invention provides a genetically engineered bacterium, which contains the recombinant vector described in the previous embodiment or the recombinant vector constructed by the construction method described in the previous embodiment.
In a sixth aspect, an embodiment of the present invention provides a method for constructing a genetically engineered bacterium according to the previous embodiment, which includes transforming the recombinant vector according to the previous embodiment or the recombinant vector constructed by the construction method according to the previous embodiment into a bacterium to obtain the genetically engineered bacterium.
In a seventh aspect, embodiments of the present invention provide a method for preparing a proteinase K mutant according to the previous embodiments, comprising: culturing the genetically engineered bacterium described in the previous examples or the genetically engineered bacterium constructed by the construction method described in the previous examples to induce expression of the proteinase K mutant; or, on the basis of the amino acid sequence shown in SEQ ID No.1, at least one of the following mutations is made: F371C and T373P.
In an eighth aspect, the present embodiment provides an application of the proteinase K mutant as described in the previous embodiment or the genetically engineered bacterium constructed by the construction method as described in the previous embodiment in protein hydrolysis or preparation of a product for protein hydrolysis.
The invention has the following beneficial effects:
compared with wild proteinase K, the proteinase K mutant provided by the invention has better enzyme activity and provides a way for proteinase K application and research.
In addition, the invention also provides the construction of the proteinase K mutant high-copy expression vector, improves the screening power of high-expression thalli, reduces the operation of a large number of screening bacteria, and indicates the direction for the industrial production route of proteinase K with application value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a three-dimensional structure of proteinase K;
FIG. 2 shows the active centers of proteinase K;
FIG. 3 shows the three-dimensional structure of proteinase K wild type and F371C/T373P mutant sites;
FIG. 4 is a plasmid map of pATG 9K-0;
FIG. 5 is a plasmid map of pATG 9-1K;
FIG. 6 is a plasmid map of pATG 9-3K;
FIG. 7 is a graph showing the results of the inhibition of high-yielding strain GS-PK on 1% casein BMMY plates;
FIG. 8 shows SDS-PAGE of the supernatants of the different transformant fermentation broths of pATG9-3K/Pichia pastoris GS.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The embodiment of the invention provides a proteinase K mutant which has at least one mutation based on an amino acid sequence shown in SEQ ID No. 1: F371C and T373P, three-dimensional structures of wild type proteinase K and F371C and T373P sites are shown in FIG. 3.
SEQ ID No.1：MRLSVLLSLLPLALGAPAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDAAMEKISGKPDHVYKNVFSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPFGTVNLLAYNNYQA。
Preferably, when F371C and T373P are present at the same time, the amino acid sequence is shown in SEQ ID No. 2: MRLSVLLSLLPLALGAPAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDAAMEKISGKPDHVYKNVFSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGLARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYYSSRDGNGHGTHCAGTVGSRTYGVAKKTQLFGVKVLDDNGSGQYSTIIAGMDFVASDKNNRNCPKGVVASLSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGASDRYDRRSSFSNYGSVLDIFGPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGKTTAASACRYIADTANKGDLSNIPCGPVNLLAYNNYQA.
An embodiment of the present invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding a proteinase K mutant as described in the previous embodiment.
Preferably, the sequence of the isolated nucleic acid is as shown in SEQ ID No.3, 5'-atgcgcctgagcgtgctgctgagcctgctgccgctggcgctgggcgcgccggcggtggaacagcgcagcgaagcggcgccgctgattgaagcgcgcggcgaaatggtggcgaacaaatatattgtgaaatttaaagaaggcagcgcgctgagcgcgctggatgcggcgatggaaaaaattagcggcaaaccggatcatgtgtataaaaacgtgtttagcggctttgcggcgaccctggatgaaaacatggtgcgcgtgctgcgcgcgcatccggatgtggaatatattgaacaggatgcggtggtgaccattaacgcggcgcagaccaacgcgccgtggggcctggcgcgcattagcagcaccagcccgggcaccagcacctattattatgatgaaagcgcgggccagggcagctgcgtgtatgtgattgataccggcattgaagcgagccatccggaatttgaaggccgcgcgcagatggtgaaaacctattattatagcagccgcgatggcaacggccatggcacccattgcgcgggcaccgtgggcagccgcacctatggcgtggcgaaaaaaacccagctgtttggcgtgaaagtgctggatgataacggcagcggccagtatagcaccattattgcgggcatggattttgtggcgagcgataaaaacaaccgcaactgcccgaaaggcgtggtggcgagcctgagcctgggcggcggctatagcagcagcgtgaacagcgcggcggcgcgcctgcagagcagcggcgtgatggtggcggtggcggcgggcaacaacaacgcggatgcgcgcaactatagcccggcgagcgaaccgagcgtgtgcaccgtgggcgcgagcgatcgctatgatcgccgcagcagctttagcaactatggcagcgtgctggatatttttggcccgggcaccagcattctgagcacctggattggcggcagcacccgcagcattagcggcaccagcatggcgaccccgcatgtggcgggcctggcggcgtatctgatgaccctgggcaaaaccaccgcggcgagcgcgtgccgctatattgcggataccgcgaacaaaggcgatctgagcaacattccgtgcggcccggtgaacctgctggcgtataacaactatcaggcg-3'.
An embodiment of the invention provides a recombinant vector comprising an isolated nucleic acid as described in any of the previous embodiments.
The embodiment of the invention provides a method for constructing a recombinant vector according to the previous embodiment, which comprises the following steps: inserting the separated nucleic acid into a vector, and constructing to obtain a single copy recombinant vector.
Preferably, the construction method further comprises: inserting the isolated nucleic acid or the expression cassette thereof into the single copy recombinant vector to obtain a double copy recombinant vector.
Preferably, the construction method further comprises inserting the isolated nucleic acid or expression cassette thereof into the double-copy recombinant vector to obtain a three-copy recombinant vector.
Preferably, the construction method of the vector is as follows: setting 6 groups of primer pairs according to the Golden gate assembly principle by taking pPIC9k as a basic skeleton, cloning to obtain 6 carrier fragments, and fusing and recombining the 6 carrier fragments to obtain the carrier;
wherein the plurality of primer pairs comprise primer pairs 1 to 6, and the sequences of the primer pairs 1 to 6 are shown as SEQ ID No.4 to 15 in sequence.
The embodiment of the invention provides a genetically engineered bacterium which contains the recombinant vector described in the previous embodiment or the recombinant vector constructed by the construction method described in any of the previous embodiments.
The embodiment of the invention provides a construction method of genetically engineered bacteria, which comprises the step of transforming the recombinant vector according to the previous embodiment or the recombinant vector constructed by the construction method according to any of the previous embodiments into bacteria to obtain the genetically engineered bacteria.
Preferably, the construction method further comprises screening the obtained transformant after transformation: inoculating the transformant into a flat plate containing antibiotics, and screening to obtain the high-expression genetically engineered bacteria based on the size of a hydrolysis ring.
Preferably, the plate is a BMMY plate containing 0.5% -2% casein.
The embodiment of the present invention provides a method for preparing the proteinase K mutant according to any of the preceding embodiments, comprising: culturing the genetically engineered bacterium described in the previous examples or the genetically engineered bacterium constructed by the construction method described in any of the previous examples to induce expression of the proteinase K mutant; or, on the basis of the amino acid sequence shown in SEQ ID No.1, at least one of the following mutations is made: F371C and T373P.
In addition, the embodiment of the invention also provides application of the proteinase K mutant or the genetically engineered bacterium constructed by the construction method in any embodiment in protein hydrolysis or preparation of a product for protein hydrolysis.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
A proteinase K mutant has the amino acid sequence shown in SEQ ID No.2 and the nucleic acid sequence shown in SEQ ID No. 3.
The preparation method of the proteinase K mutant comprises the following steps.
1. Construction of the expression vector.
1.1 construction of Single copy expression vectors.
The pPIC9k is used as a basic skeleton, and corresponding primers are designed according to the Golden gate assembly principle, see Table 1, and six framework fragments named as Frag1, frag2, frag3, frag4, frag5 and Frag6 are obtained by cloning with P202-2 high-fidelity polymerase of Nanjing giant biotechnology Co., ltd.
TABLE 1 primer sequences
Remarks: f is an upstream primer, and R is a downstream primer.
The DNA fragment with higher purity is recovered from the fragment gel obtained by cloning, and the fragment is used as a template to fuse the fragment 1 with the fragment 2, the fragment 3 with the fragment 4 and the fragment 5 with the fragment 6 by overlap extension PCR. The overlap extension PCR system is shown in Table 2.
TABLE 2 PCR System
System of Usage amount
Fragment 1 2μl
Fragment 2 2μl
2×ATG Proofast MIX 25μl
Primer F 1.5μl
Primer R 1.5μl
ddH 2 O Supplement to 50 μl
Reference is made to the protocol of P202-2 Hi-Fi polymerase, inc. of Biotechnology, cytomer GmbH, nanj.
The obtained fragment gel is recovered to obtain a fusion fragment, the fusion fragment is subjected to recombination connection through a recombination cloning kit C101 of Nanjing great worker biotechnology limited company to obtain a recombination vector connection product pATG9K-0, a plasmid map is shown in figure 4, the fusion fragment is converted into XL10 competence, an Amp resistance gene plate is coated, a positive transformant is screened, and plasmids are extracted for nucleic acid electrophoresis verification.
A linearized vector was obtained by restriction endonuclease Not I and Cop I cleavage of pATG9K-0, and simultaneously a protease K gene linearized fragment Frag7 was obtained by the primers shown in Table 3.
TABLE 3 primers
PRK-F ATGCGTTTATCTGTTTTGCTTTCCTC
PRK-R CTGAAACATATTGGCCAAGCAGTGATG
The single copy vector pATG9-1K is obtained through the recombination connection of the obtained vector fragment and the gene fragment by a recombination cloning kit C111 of Nanjing great worker biotechnology limited company, the plasmid map is shown in figure 5, the plasmid map is transformed into XL10 competence, an Amp resistance gene plate is coated, positive transformants are screened, verified by a verification primer, and sequencing verification is carried out.
1.2 double copy expression vector construction
After successful verification of the recombinant vector pATG9-1K, the pATG9-1K plasmid was extracted, linearized by the restriction endonuclease BamH I to obtain a linear vector fragment Frag8, and digested by BamH I and Bgl II to pATG9-K1 plasmid to obtain an expression cassette of proteinase K (AOX 1 promoter+proteinase K+AOX1 terminator) fragment Frag9.
The two fragments of Frag8 and Frag9 were subjected to gel recovery to obtain high purity DNA fragments, which were ligated overnight at 16℃by T4DNA ligase according to the ligation system as shown in Table 4.
Table 4 connection system
10×T4 DNA Ligase Buffer 2μl
DNA fragment 1μl
Vector DNA 0.5μl
T4 DNA Ligase 0.5μl
ddH 2 O To 20μl
The ligation products are transformed into XL10 competence, amp resistance gene plates are coated, positive transformants are screened, the construction success of a double copy vector pATG9-2K is verified by using verification primers, and sequencing verification is carried out.
1.3 construction of multicopy expression vectors
The restriction endonuclease BamH I was further linearized to obtain the linear vector fragment Frag10 in a similar manner to step 1.2, frag10 and Frag9 were ligated by T4DNA ligase to obtain the ligation product of recombinant vector pATG9-3K, plasmid map shown in FIG. 6.
1.4 screening of strains
The ligation product was transferred to E.coli XL10 competent, and screened by plating on LB plates containing 100. Mu.g/ml Amp resistance to obtain pATG9-3K/XL10 strain.
Example 2
A proteinase K mutant and a method for preparing the same are substantially the same as in example 1, except that the screening steps of the strain are different, in this example, the strain is screened as follows: linearizing the pATG9-3K plasmid vector, electrically transferring the plasmid vector into pichia pastoris GS115 competent cells, and screening by a casein BMMY flat plate containing G418 with different concentrations and 1% to obtain a high-copy high-expression strain, wherein the method specifically comprises the following steps:
performing enzyme tangentially on the pATG9-3K plasmid vector by SacI, cutting off a target fragment by 0.8% agarose nucleic acid electrophoresis, and recovering and purifying the gel;
the purified gene fragment was transformed into pichia pastoris GS115 competent cells by electrotransformation, spread on MD plates, cultured at 28℃for 2-3 days, the Pichia pastoris transformants on MD plates were transferred to 1% casein BMMY plates containing different concentrations (50. Mu.g/ul-500. Mu.g/ul) of G418 for selection, high copy number Pichia pastoris recombinant transformants were selected according to the size of the hydrolytic circle, and the strain with the largest hydrolytic circle was designated GS-PK, see FIG. 7.
Single colony colonies were inoculated into 25ml BMGY medium and cultured overnight at 28℃for 16-20h to an OD600 of 2-6. The bacterial solution was further transferred to a 50ml centrifuge tube, the supernatant was removed by centrifugation, and then the bacterial cells were resuspended in 100ml of BMMY medium, 1ml of methanol was added, and the cells were cultured at 28℃for 120 hours. 1ml of 100% methanol was added every 24 hours;
4. after the fermentation culture is finished, the supernatant of the fermentation broth is collected by centrifugation to obtain a proteinase K crude enzyme solution, the molecular weight, purity and yield of the target protein are detected by SDS-PAGE, and the protein concentration is tested by a Bradford method, and the result is shown in figure 8.
Example 3
The activity of the proteinase K mutants provided in example 1 was verified.
Method
Primers were designed for F371C and T373P, respectively, as shown in Table 5.
Table 5 primers
371/373-F CCTGTCCAACATTCCCTGTGGCCCGGTCAATTTATTGGC
371/373-R GCCAATAAATTGACCGGGCCACAGGGAATGTTGGACAGG
The pUC-PRK plasmid is used as a template, F371C-F/F371C-R primer is used for cloning to obtain a fragment Frag11, a target fragment is recovered through agarose nucleic acid electrophoresis, the target fragment is connected to a pMD18T vector through Solution I and is converted into JM109 competence, a transformant is brushed and selected in an Amp-resistant LB plate, the transformant is picked and transferred into an LB liquid medium for culturing for 8-12h for sequencing verification, and the named pUC-Mut-1 with successful first site mutation is obtained.
And then pUC-Mut-1 is used as a template, a fragment 12 is obtained by cloning with a T373P-F/T373P-R primer, and a pUC-Mut-12 recombinant vector is constructed in the same way, so that an F371C/T373P double mutation point proteinase K gene DNA fragment is obtained.
The pUC-Mut-12 vector is used as a template to obtain pATG9-3K by the experimental method Mut-12 And (3) carrying out enzyme tangential treatment on the recombinant vector by SacI, and recovering and purifying the recombinant vector by gel to obtain the target fragment.
The purified target fragment is electrically transformed into pichia pastoris GS115 competent cells, coated on an MD plate and cultured for 2-3 days at 28 ℃, and the pichia pastoris transformant on the MD plate is transferred to a 1% casein BMMY plate with low concentration G418 to screen mutant strains.
And (3) fermenting and inducing the mutant strain, and centrifugally collecting proteinase K crude enzyme liquid.
Taking the obtained proteinase K crude enzyme to test the activity of the proteinase K crude enzyme: ultraviolet spectrophotometry is to use casein as substrate, and measure 1 mu g L-tyrosine produced by hydrolyzing casein by proteinase K in 0.02mol/L Tris-HCl buffer solution with pH of 8.0 at 55 ℃ at 275nm wavelength for 1 min.
The results are shown in Table 6.
TABLE 6 protease K enzyme Activity test
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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Ser Thr Arg Ser Ile Ser Gly Thr Ser Met Ala Thr Pro His Val Ala
325 330 335
Gly Leu Ala Ala Tyr Leu Met Thr Leu Gly Lys Thr Thr Ala Ala Ser
340 345 350
Ala Cys Arg Tyr Ile Ala Asp Thr Ala Asn Lys Gly Asp Leu Ser Asn
355 360 365
Ile Pro Cys Gly Pro Val Asn Leu Leu Ala Tyr Asn Asn Tyr Gln Ala
370 375 380
<210> 3
<211> 1152
<212> DNA
<213> artificial sequence
<400> 3
atgcgcctga gcgtgctgct gagcctgctg ccgctggcgc tgggcgcgcc ggcggtggaa 60
cagcgcagcg aagcggcgcc gctgattgaa gcgcgcggcg aaatggtggc gaacaaatat 120
attgtgaaat ttaaagaagg cagcgcgctg agcgcgctgg atgcggcgat ggaaaaaatt 180
agcggcaaac cggatcatgt gtataaaaac gtgtttagcg gctttgcggc gaccctggat 240
gaaaacatgg tgcgcgtgct gcgcgcgcat ccggatgtgg aatatattga acaggatgcg 300
gtggtgacca ttaacgcggc gcagaccaac gcgccgtggg gcctggcgcg cattagcagc 360
accagcccgg gcaccagcac ctattattat gatgaaagcg cgggccaggg cagctgcgtg 420
tatgtgattg ataccggcat tgaagcgagc catccggaat ttgaaggccg cgcgcagatg 480
gtgaaaacct attattatag cagccgcgat ggcaacggcc atggcaccca ttgcgcgggc 540
accgtgggca gccgcaccta tggcgtggcg aaaaaaaccc agctgtttgg cgtgaaagtg 600
ctggatgata acggcagcgg ccagtatagc accattattg cgggcatgga ttttgtggcg 660
agcgataaaa acaaccgcaa ctgcccgaaa ggcgtggtgg cgagcctgag cctgggcggc 720
ggctatagca gcagcgtgaa cagcgcggcg gcgcgcctgc agagcagcgg cgtgatggtg 780
gcggtggcgg cgggcaacaa caacgcggat gcgcgcaact atagcccggc gagcgaaccg 840
agcgtgtgca ccgtgggcgc gagcgatcgc tatgatcgcc gcagcagctt tagcaactat 900
ggcagcgtgc tggatatttt tggcccgggc accagcattc tgagcacctg gattggcggc 960
agcacccgca gcattagcgg caccagcatg gcgaccccgc atgtggcggg cctggcggcg 1020
tatctgatga ccctgggcaa aaccaccgcg gcgagcgcgt gccgctatat tgcggatacc 1080
gcgaacaaag gcgatctgag caacattccg tgcggcccgg tgaacctgct ggcgtataac 1140
aactatcagg cg 1152
<210> 4
<211> 53
<212> DNA
<213> artificial sequence
<400> 4
cctttcgtct ttggatgtta gatctagatc ttgagataaa tttcacgttt aaa 53
<210> 5
<211> 44
<212> DNA
<213> artificial sequence
<400> 5
gatccttcaa ctcagcaaaa gttcgcctgc aggtcggcat cacc 44
<210> 6
<211> 44
<212> DNA
<213> artificial sequence
<400> 6
ggtgatgccg acctgcaggc gaacttttgc tgagttgaag gatc 44
<210> 7
<211> 58
<212> DNA
<213> artificial sequence
<400> 7
tgtgaaatag acgcagatcg ggagtcgaca attggtttga ctaattccat aatctgtt 58
<210> 8
<211> 58
<212> DNA
<213> artificial sequence
<400> 8
aacagattat ggaattagtc aaaccaattg tcgactcccg atctgcgtct atttcaca 58
<210> 9
<211> 52
<212> DNA
<213> artificial sequence
<400> 9
ctttgtcacc attttgacga acattgtcga ctccgcttcc tcgctcactg ac 52
<210> 10
<211> 52
<212> DNA
<213> artificial sequence
<400> 10
gtcagtgagc gaggaagcgg agtcgacaat gttcgtcaaa atggtgacaa ag 52
<210> 11
<211> 45
<212> DNA
<213> artificial sequence
<400> 11
catcttcccg acaacgcgcc ggaccggcgg ccgcgaatta attcg 45
<210> 12
<211> 45
<212> DNA
<213> artificial sequence
<400> 12
cgaattaatt cgcggccgcc ggtccggcgc gttgtcggga agatg 45
<210> 13
<211> 50
<212> DNA
<213> artificial sequence
<400> 13
cgagaaaaga gaggctgaag cttaccggcc gccggggcct gccaccatac 50
<210> 14
<211> 50
<212> DNA
<213> artificial sequence
<400> 14
gtatggtggc aggccccggc ggccggtaag cttcagcctc tcttttctcg 50
<210> 15
<211> 53
<212> DNA
<213> artificial sequence
<400> 15
tttaaacgtg aaatttatct caagatctag atctaacatc caaagacgaa agg 53

Claims (15)

1. Proteinase K mutant, characterized in that it has at least one mutation on the basis of the amino acid sequence shown in SEQ ID No. 1: F371C and T373P.
2. Proteinase K mutant according to claim 1, characterized in that its amino acid sequence is shown in SEQ ID No. 2.
3. An isolated nucleic acid comprising a nucleic acid sequence encoding the proteinase K mutant of claim 1 or 2.
4. The isolated nucleic acid of claim 3, wherein the sequence of the isolated nucleic acid is set forth in SEQ ID No. 3.
5. A recombinant vector comprising the isolated nucleic acid of claim 3 or 4.
6. The method for constructing a recombinant vector according to claim 5, comprising: inserting the separated nucleic acid into a vector, and constructing to obtain a single copy recombinant vector.
7. The method for constructing a recombinant vector according to claim 6, further comprising: inserting the isolated nucleic acid or the expression cassette thereof into the single copy recombinant vector to obtain a double copy recombinant vector.
8. The method of claim 6, further comprising inserting the isolated nucleic acid or the expression cassette thereof into the double-copy recombinant vector to obtain a three-copy recombinant vector.
9. The method for constructing a recombinant vector according to claim 6, wherein the method for constructing the vector comprises the steps of: setting 6 groups of primer pairs according to the Golden gate assembly principle by taking pPIC9k as a basic skeleton, cloning to obtain 6 carrier fragments, and fusing and recombining the 6 carrier fragments to obtain the carrier;
the 6 groups of primer pairs comprise primer pairs 1-6, and sequences of the primer pairs 1-6 are sequentially shown as SEQ ID No. 4-15.
10. A genetically engineered bacterium comprising the recombinant vector according to claim 5 or the recombinant vector constructed by the construction method according to any one of claims 6 to 9.
11. The method for constructing a genetically engineered bacterium according to claim 10, comprising transforming the recombinant vector according to claim 5 or the recombinant vector constructed by the construction method according to any one of claims 6 to 9 into a bacterium to obtain the genetically engineered bacterium.
12. The method for constructing genetically engineered bacteria of claim 11, further comprising screening the transformed obtained transformants: inoculating the transformant into a flat plate containing antibiotics, and screening to obtain the high-expression genetically engineered bacteria based on the size of a hydrolysis ring.
13. The method for constructing genetically engineered bacteria of claim 12, wherein the plate is a BMMY plate containing 0.5% -2% casein.
14. The method for producing a proteinase K mutant according to claim 1 or 2, which comprises: culturing the genetically engineered bacterium of claim 10 or the genetically engineered bacterium constructed by the construction method of any one of claims 11 to 13 to induce expression of the proteinase K mutant; or alternatively, the first and second heat exchangers may be,
based on the amino acid sequence shown in SEQ ID No.1, at least one of the following mutations is made: F371C and T373P.
15. The use of the proteinase K mutant according to claim 1 or 2 or the genetically engineered bacterium according to claim 10 or the genetically engineered bacterium constructed by the construction method according to any one of claims 11 to 13 for hydrolyzing proteins or for preparing products for hydrolyzing proteins.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996028556A2 (en) * 1995-03-09 1996-09-19 The Procter & Gamble Company Proteinase k variants having decreased adsorption and increased hydrolysis
CN102839165A (en) * 2012-09-26 2012-12-26 金普诺安生物科技（苏州）有限公司 Gene mutation type recombined protease K and industrialized production method thereof
CN104480027A (en) * 2015-01-04 2015-04-01 青岛蔚蓝生物集团有限公司 Aspergillus niger strain of high-yield protease K and application of aspergillus niger strain
CN113215138A (en) * 2021-06-02 2021-08-06 武汉瀚海新酶生物科技有限公司 Proteinase K mutant with improved thermal stability
CN113337492A (en) * 2021-06-02 2021-09-03 武汉瀚海新酶生物科技有限公司 Protease K heat-resistant mutant
CN113481225A (en) * 2021-07-23 2021-10-08 武汉瀚海新酶生物科技有限公司 Construction and application of protease K high-expression engineering strain

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* Cited by examiner, † Cited by third party
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WO1996028556A2 (en) * 1995-03-09 1996-09-19 The Procter & Gamble Company Proteinase k variants having decreased adsorption and increased hydrolysis
CN102839165A (en) * 2012-09-26 2012-12-26 金普诺安生物科技（苏州）有限公司 Gene mutation type recombined protease K and industrialized production method thereof
CN104480027A (en) * 2015-01-04 2015-04-01 青岛蔚蓝生物集团有限公司 Aspergillus niger strain of high-yield protease K and application of aspergillus niger strain
CN113215138A (en) * 2021-06-02 2021-08-06 武汉瀚海新酶生物科技有限公司 Proteinase K mutant with improved thermal stability
CN113337492A (en) * 2021-06-02 2021-09-03 武汉瀚海新酶生物科技有限公司 Protease K heat-resistant mutant
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