CN113913412B - Proteinase K mutant and its preparing process - Google Patents
Proteinase K mutant and its preparing process Download PDFInfo
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- CN113913412B CN113913412B CN202111193669.3A CN202111193669A CN113913412B CN 113913412 B CN113913412 B CN 113913412B CN 202111193669 A CN202111193669 A CN 202111193669A CN 113913412 B CN113913412 B CN 113913412B
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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)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
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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
- 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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- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/102—Plasmid DNA for yeast
Abstract
The invention discloses a proteinase K mutant and a preparation method thereof, belonging to the technical field of protein engineering, wherein the mutant comprises the following components: the 44 th amino acid of the wild type proteinase K shown in SEQ ID NO.1 is mutated, in particular the 44 th alanine is mutated into serine. The invention is based on the crystal structure of proteinase K, and the proteinase K is modified by protein engineering, site-directed mutagenesis and other techniques to obtain mutant A44S shown in SEQ ID NO.3 for the first time. The research shows that the specific enzyme activity of the mutant A44S is improved by about 21 percent compared with that of a wild type, so that the application cost of proteinase K is reduced, the industrial application value of the enzyme in various industries such as washing, feed, food and the like is improved, and an important clue is provided for the research on the catalytic mechanism of the enzyme.
Description
Technical Field
The invention belongs to the technical field of protein engineering, and particularly relates to a proteinase K mutant and a preparation method thereof.
Background
Proteinase K is a serine proteinase from Lin Bashi C.albumins (Tritirachium album limber) with very high enzyme activity and broad substrate spectrum, and has high efficient cutting ability to natural protein under broad pH conditions, especially alkaline conditions, and the proteinase is better than the carboxyl terminal peptide bond of aliphatic amino acid and aromatic amino acid, and can preferentially decompose the ester bond and peptide bond adjacent to the C terminal of hydrophobic amino acid, sulfur-containing amino acid and aromatic amino acid. In scientific research, the enzyme is widely applied to the extraction process of nucleic acid and is an important tool enzyme. With the development of nucleic acid detection technology, proteinase K is taken as degradation protein, important enzymes of RT-qPCR templates are extracted, and market demand is increased. Meanwhile, the enzyme has good resistance to SDS, urea and the like, so that the enzyme has good application effects in the fields of washing industry, feed industry, sewage treatment, papermaking, food and the like.
Heterologous expression of proteinase K in E.coli cells was achieved for the first time by Gunkel et al in 1989, and its basic properties were studied. With the advancement of technology, the crystal structure of proteinase K was resolved and the enzyme was found to be a typical serine protease. The enzyme has two Ca bound thereto 2+ Has important promoting effect on the activity and the thermal stability. However, the expression level of the enzyme in Lin Bashi candida albicans is very low, the enzyme is difficult to apply to actual production, and the enzyme has high activity under alkaline conditions, can endure detergent with a certain concentration and has good application prospect in the washing industry, but the activity of the enzyme has a certain gap compared with that of the protease for washing commercialized abroad. It is therefore important how proteinase K can be engineered to increase its expression and enzymatic activity.
Disclosure of Invention
The invention aims to provide a proteinase K mutant, which is based on the crystal structure of proteinase K, and the proteinase molecule is modified by a rational design method, so that the specific activity of the proteinase K mutant is improved, and the proteinase K mutant can be possibly applied to the washing industry.
It is an object of the present invention to provide a proteinase K mutant, which is: the 44 th amino acid of the wild type proteinase K is mutated, wherein the amino acid sequence of the wild type proteinase K is shown as SEQ ID NO. 1.
Further, the nucleotide sequence for encoding the wild type proteinase K is shown as SEQ ID NO. 2.
Further, the mutants are: alanine at position 44 of wild-type proteinase K was mutated to serine.
Further, the amino acid sequence of the mutant is shown as SEQ ID NO. 3.
Further, the nucleotide sequence of the encoding mutant is shown as SEQ ID NO. 4.
It is a second object of the present invention to provide a vector comprising the above-described nucleotide encoding a mutant.
It is a further object of the present invention to provide a cell comprising the vector.
The fourth object of the present invention is to provide a method for producing a proteinase K mutant, comprising the steps of:
step 1, obtaining the nucleotide of the coding mutant shown as SEQ ID NO. 4;
step 2, fusing the nucleotide obtained in the step 1 with an expression vector, constructing a recombinant expression vector, and converting the recombinant expression vector into a host cell Pichia pastoris GS 115;
and step 3, inducing host cells containing the recombinant expression vector to express, and separating and purifying to obtain the proteinase K mutant protein.
Further, in the step 1, the nucleotide sequence of the encoding wild type proteinase K shown as SEQ ID NO.2 is used as a template, and the nucleotide of the encoding mutant shown as SEQ ID NO.4 is obtained by adopting a site-directed mutagenesis method.
Further, the method for site-directed mutagenesis comprises: the nucleotide sequence of the coded wild protease K shown in SEQ ID NO.2 is used as a template, and F primer, A44S-R primer, A44S-F primer and R primer are respectively used as primer pairs for PCR amplification to obtain two sections of PCR products with mutation points as two ends; and then, taking two sections of PCR products as templates, taking F primer and R primer as primers to carry out PCR, and connecting the primers to obtain the nucleotide of the coding mutant shown as SEQ ID NO. 4.
Further, the nucleotide sequence of the F primer is shown as SEQ ID NO. 5; the nucleotide sequence of the A44S-R primer is shown in SEQ ID NO. 6; the nucleotide sequence of the A44S-F primer is shown in SEQ ID NO. 7; the nucleotide sequence of the R primer is shown as SEQ ID NO. 8.
It is a fifth object of the present invention to provide a method for increasing proteinase K activity, said method comprising: alanine 44 of wild-type proteinase K as shown in SEQ ID NO.1 was mutated to serine.
Compared with the prior art, the invention has the beneficial effects that: aiming at the problem of relatively low activity of the existing proteinase K, the invention selects a plurality of amino acid sites near the active center of proteinase K based on the crystal structure of proteinase K, and modifies proteinase K through techniques such as protein engineering, site-directed mutagenesis and the like to obtain mutant A44S with 44 th alanine mutated into serine for the first time, and the amino acid sequence is shown in SEQ ID NO. 3. The research shows that the specific enzyme activity of the mutant A44S is improved by about 21 percent compared with that of a wild type, so that the application cost of proteinase K is reduced, the industrial application value of the enzyme in various industries such as washing, feed, food and the like is improved, and an important clue is provided for the research on the catalytic mechanism of the enzyme.
Drawings
FIG. 1 shows the SDS-PAGE results of wild-type proteinase K and its mutant A44S according to example 3 of the present invention, wherein M is Marker, lane 1 is wild-type proteinase K and lane 2 is mutant A44S.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden on the person of ordinary skill in the art based on embodiments of the present invention, are within the scope of the present invention.
Example 1
1. Acquisition of A44S mutant
The nucleotide sequence of the code wild proteinase K shown in SEQ ID No.2 is used as a template, F primer, A44S-R primer, A44S-F primer and R primer are respectively used as primer pairs, and conventional PCR amplification is respectively carried out to obtain two sections of PCR products with mutation points as two ends. And performing conventional PCR amplification by taking two sections of PCR products as templates and taking F primer and R primer as primers, and connecting the primers to obtain the coding gene of the mutant A44S shown in SEQ ID NO. 4. The primer sequences used were as follows: f primer: gtagaattaagatcccgacacatggctccagccgttgaacaaagatc (SEQ ID NO. 5); A44S-R primer: ccctaccttcaaattctggatgagaAGActcaata (SEQ ID NO. 6); A44S-F primer: gtgtttacgttattgatactggtattgagTCTtctcatccag (SEQ ID NO. 7); r primer: gtctaaggcgaattaattcgcacttaatggtgatggtgatggtgagctt (SEQ ID NO. 8),
wherein the uppercase parts in the A44S-R primer and the A44S-F primer are mutation sites.
2. Recombinant expression vector construction
Pichia pastoris expression vector pHBM905BDM is digested with NotI and CpoI, recovered and mixed with the encoding gene fragment of mutant A44S obtained by PCR amplification, and treated with 0.1U of T5 exonuclease on ice-water mixture for 3-5min. And then transferring the strain into E.coli XL10-Gold competent cells. The transformation solution was coated on LB plate containing ampicillin (100 mg/L), the plasmid was extracted, and the constructed recombinant plasmid was verified by colony PCR and named pHBM905BDM-A44S. Sequencing work was done by Shanghai workers.
The primers used for colony PCR were as follows:
colony PCR-F primer: gcatcttctgctttggctgctc (shown as SEQ ID NO. 9)
Colony PCR-R primer: cgagataggctgatcaggagcaag (shown as SEQ ID NO. 10)
EXAMPLE 2 transformation and selection of recombinant proteinase K mutant plasmids in Pichia pastoris
(1) The recombinant proteinase K mutant expression vector pHBM905BDM-A44S prepared in the example is transformed into pichia pastoris GS115 competent cells according to a conventional method, and specifically comprises the following steps: the recombinant proteinase K mutant vector pHBM905BDM-A44S is digested by using the restriction enzyme SalI, then glue is recovered, 10 mu L of linear fragments are evenly mixed with 100 mu L of GS115 yeast competent cells, the mixture is transferred into an ice-precooled electrorotating cup (the gap between two electrodes is 2 mm), and the electrorotating cup is placed into an electrorotating instrument for electric shock, and the electric shock parameters are as follows: 1500V,25 μF,200Ω; then 200 mu L of 1M sorbitol is added into the electric rotating cup, 200 mu L of MD culture medium is added, the mixture is uniformly mixed and then transferred into a 1.5mL EP tube, and the mixture is subjected to shaking table activation for 2 hours at 28 ℃; the bacterial suspension was centrifuged to remove the supernatant, resuspended in 80. Mu.L of MD medium, and plated onto MD plates (glucose 20.0g/L, (NH) 4 ) 2 SO 4 10.0g/L, YNB 13.4g/L, agar 2%,500 XBiotin 0.02%,2 mL), were inverted in a 28℃incubator and cultured until single colonies appeared.
(2) The single colony of the yeast obtained by transformation is simultaneously time-point on a BMGY plate and an MD plate, and the same single colony is marked consistently; after about 36-48 hours, the colony corresponding points on the BMGY plate are reached to a 1% casein BMMY plate, and the marks are kept consistent; 200 mu L of methanol is added to filter paper every 12 hours for induction; observing hydrolysis rings on the casein plate after 48-60 hours of induction, wherein single bacterial colony corresponding to the hydrolysis rings is proteinase K recombinant bacterial colony; the recombinant colony corresponding to the hydrolysis circle is cultured to OD by YPD culture medium 600 Taking 500 mu L of bacterial liquid and equal volume of 50% glycerol to mix uniformly in a freeze-drying tube, and storing in a refrigerator at-80 ℃.
EXAMPLE 3 shake flask expression and enzymatic Property determination of wild-type proteinase K and its mutants in Pichia pastoris
(1) The wild proteinase K and A44S mutant expression strains are respectively inoculated in l00mL BMGY culture medium and shake-cultured at 28 ℃ until OD 600 6-10. After that, the cells were collected by centrifugation at 3000rpm for 5min, and resuspended in 50mL of BMMY medium. Shaking culture at 28deg.C, adding methanol to final concentration of 1% every 12 hr, and continuously inducing for about 120 hr, and taking samples periodically. After 120h of induction, the induced bacterial solution was centrifuged (4000 rpm,5 min), the supernatant was filtered with a 0.22 μm filter, and the filtrate was stored in a sterile 50mL centrifuge tube and kept in a refrigerator at 4 ℃.
(2) Protein ultrafiltration is changed, the filtered protein is transferred into a 10kD ultrafiltration tube, centrifuged at 4000rpm and 4 ℃, and changed with 3 times volume of PBS buffer, and finally concentrated proteinase K is placed at 4 ℃ for storage. The wild type proteinase K and the A44S mutant are detected by SDS-PAGE gel respectively, and the detection result is shown in figure 1, wherein a lane 1 is the wild type proteinase K, and a lane 2 is the A44S mutant. And drawing a standard curve by using the Bradford protein concentration determination kit, and calculating the corresponding protein concentration.
(3) Enzyme activity determination: enzyme activity is defined as the amount of enzyme that hydrolyzes casein to 1 μg tyrosine in 1min under certain temperature and pH conditions as one enzyme activity unit, denoted as U. The determination method mainly refers to national standard BG/T28715-2012, but the reaction system is reduced. The reaction process is as follows: 100. Mu.L of enzyme solution was added to a 1.5mL EP tube, preheated in a 55℃water bath for 3min, 100. Mu.L of the same preheated 1% casein solution was added, shaken well, then in a 55℃water bath for 10min, 200. Mu.L of trichloroacetic acid solution was added, and shaken well (blank control was added trichloroacetic acid first and then casein solution). Take out and cool to room temperature and centrifuge at 12000rpm for 10min. 100. Mu.L of the supernatant was taken, 500. Mu.L of sodium carbonate solution was added, and diluted furin reagent (commercial Folin: ddH) 2 O=1: 2) 100. Mu.L was developed at 40℃for 20min, and absorbance was measured at 680nm using a microplate reader.
The protein concentrations and the enzyme activity measurements of the wild type and the mutant are shown in Table 1.
TABLE 1 concentration of wild-type proteinase K and mutants thereof and results of specific enzyme activity measurement
As can be seen from the comprehensive measurement results of FIG. 1 and Table 1, the expression level of the proteinase K mutant A44S prepared by the method is slightly reduced compared with that of the wild proteinase K, but the enzyme activity is obviously improved by about 21%, namely, the mutant A44S is obtained by mutating the wild proteinase K, so that the application cost of the mutant A44S is reduced, the industrial application value of the enzyme in various industries such as washing, feed, food and the like is improved, and an important clue is provided for the catalytic mechanism research of the enzyme.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
Sequence listing
<110> university of Hubei
<120> proteinase K mutant and method for preparing the same
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 279
<212> PRT
<213> Lin Bashi white candida (Tritirachium album limber)
<400> 1
Ala Ala Gln Thr Asn Ala Pro Trp Gly Leu Ala Arg Ile Ser Ser Thr
1 5 10 15
Ser Pro Gly Thr Ser Thr Tyr Tyr Tyr Asp Glu Ser Ala Gly Gln Gly
20 25 30
Ser Cys Val Tyr Val Ile Asp Thr Gly Ile Glu Ala Ser His Pro Glu
35 40 45
Phe Glu Gly Arg Ala Gln Met Val Lys Thr Tyr Tyr Ala Ser Ser Arg
50 55 60
Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr Val Gly Ser Arg
65 70 75 80
Thr Tyr Gly Val Ala Lys Lys Thr Gln Leu Phe Gly Val Lys Val Leu
85 90 95
Asp Asp Asn Gly Ser Gly Gln Tyr Ser Thr Ile Ile Ala Gly Met Asp
100 105 110
Phe Val Ala Ser Asp His Asn Asn Arg Asn Cys Pro Lys Gly Val Val
115 120 125
Ala Ser Leu Ser Leu Gly Gly Gly Tyr Ser Ser Ser Val Asn Ser Ala
130 135 140
Ala Ala Arg Leu Gln Ser Ser Gly Val Met Val Ala Val Ala Ala Gly
145 150 155 160
Asn Asn Asn Ala Asp Ala Arg Asn Tyr Ser Pro Ala Ser Glu Pro Ser
165 170 175
Val Cys Thr Val Gly Ala Thr Asp Arg Tyr Asp Arg Arg Ser Ser Phe
180 185 190
Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Ala Pro Gly Thr Ser Ile
195 200 205
Leu Ser Thr Trp Ile Gly Gly Ser Thr Arg Ser Ile Ser Gly Thr Ser
210 215 220
Met Ala Thr Pro His Val Ala Gly Leu Ala Ala Tyr Leu Met Thr Leu
225 230 235 240
Gly Arg Thr Thr Ala Ala Asn Ala Cys Arg Tyr Ile Ala Asp Thr Ala
245 250 255
Asn Lys Gly Asp Leu Ser Asn Ile Pro Phe Gly Thr Val Asn Leu Leu
260 265 270
Ala Tyr Asn Asn Tyr Gln Ala
275
<210> 2
<211> 840
<212> DNA
<213> Lin Bashi white candida (Tritirachium album limber)
<400> 2
gctgcacaaa ctaacgctcc atggggattg gctagaattt cttctacttc tccaggtact 60
tcaacatact actatgatga atctgcaggt cagggtagtt gtgtttacgt tattgatact 120
ggtattgagg cttctcatcc agaatttgaa ggtagggctc aaatggtgaa gacttattac 180
gcttcatcaa gagatggtaa cggtcatggt actcattgtg ctggtaccgt tggttctagg 240
acttacggtg ttgctaagaa gactcaactg tttggtgtta aggttttgga tgataatggc 300
agtggtcaat attctactat tattgcaggt atggattttg ttgcatctga tcataacaac 360
agaaactgtc caaagggtgt tgttgcttct ttgtctttgg gcggtggtta ctcttcttct 420
gtgaactctg ccgcagcccg tttgcagtct agtggtgtaa tggtcgctgt tgcagcaggt 480
aacaacaacg cagatgctag aaattactct cccgcttctg agccatctgt atgcacggtt 540
ggagccactg acagatacga tagacgttct agtttttcta actacggctc tgttcttgac 600
atttttgctc caggaacttc tattttgtct acttggattg gaggctctac aaggtctata 660
tcaggtacat ctatggctac tccacacgtt gccggtttgg ctgcctactt aatgactttg 720
ggtagaacta ctgctgctaa cgcttgcaga tatattgccg atacagctaa taagggtgat 780
ttgagtaaca ttccatttgg tactgtcaat ttgttggctt acaataacta ccaagcttaa 840
<210> 3
<211> 279
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Ala Ala Gln Thr Asn Ala Pro Trp Gly Leu Ala Arg Ile Ser Ser Thr
1 5 10 15
Ser Pro Gly Thr Ser Thr Tyr Tyr Tyr Asp Glu Ser Ala Gly Gln Gly
20 25 30
Ser Cys Val Tyr Val Ile Asp Thr Gly Ile Glu Ser Ser His Pro Glu
35 40 45
Phe Glu Gly Arg Ala Gln Met Val Lys Thr Tyr Tyr Ala Ser Ser Arg
50 55 60
Asp Gly Asn Gly His Gly Thr His Cys Ala Gly Thr Val Gly Ser Arg
65 70 75 80
Thr Tyr Gly Val Ala Lys Lys Thr Gln Leu Phe Gly Val Lys Val Leu
85 90 95
Asp Asp Asn Gly Ser Gly Gln Tyr Ser Thr Ile Ile Ala Gly Met Asp
100 105 110
Phe Val Ala Ser Asp His Asn Asn Arg Asn Cys Pro Lys Gly Val Val
115 120 125
Ala Ser Leu Ser Leu Gly Gly Gly Tyr Ser Ser Ser Val Asn Ser Ala
130 135 140
Ala Ala Arg Leu Gln Ser Ser Gly Val Met Val Ala Val Ala Ala Gly
145 150 155 160
Asn Asn Asn Ala Asp Ala Arg Asn Tyr Ser Pro Ala Ser Glu Pro Ser
165 170 175
Val Cys Thr Val Gly Ala Thr Asp Arg Tyr Asp Arg Arg Ser Ser Phe
180 185 190
Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Ala Pro Gly Thr Ser Ile
195 200 205
Leu Ser Thr Trp Ile Gly Gly Ser Thr Arg Ser Ile Ser Gly Thr Ser
210 215 220
Met Ala Thr Pro His Val Ala Gly Leu Ala Ala Tyr Leu Met Thr Leu
225 230 235 240
Gly Arg Thr Thr Ala Ala Asn Ala Cys Arg Tyr Ile Ala Asp Thr Ala
245 250 255
Asn Lys Gly Asp Leu Ser Asn Ile Pro Phe Gly Thr Val Asn Leu Leu
260 265 270
Ala Tyr Asn Asn Tyr Gln Ala
275
<210> 4
<211> 840
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
gctgcacaaa ctaacgctcc atggggattg gctagaattt cttctacttc tccaggtact 60
tcaacatact actatgatga atctgcaggt cagggtagtt gtgtttacgt tattgatact 120
ggtattgagt cttctcatcc agaatttgaa ggtagggctc aaatggtgaa gacttattac 180
gcttcatcaa gagatggtaa cggtcatggt actcattgtg ctggtaccgt tggttctagg 240
acttacggtg ttgctaagaa gactcaactg tttggtgtta aggttttgga tgataatggc 300
agtggtcaat attctactat tattgcaggt atggattttg ttgcatctga tcataacaac 360
agaaactgtc caaagggtgt tgttgcttct ttgtctttgg gcggtggtta ctcttcttct 420
gtgaactctg ccgcagcccg tttgcagtct agtggtgtaa tggtcgctgt tgcagcaggt 480
aacaacaacg cagatgctag aaattactct cccgcttctg agccatctgt atgcacggtt 540
ggagccactg acagatacga tagacgttct agtttttcta actacggctc tgttcttgac 600
atttttgctc caggaacttc tattttgtct acttggattg gaggctctac aaggtctata 660
tcaggtacat ctatggctac tccacacgtt gccggtttgg ctgcctactt aatgactttg 720
ggtagaacta ctgctgctaa cgcttgcaga tatattgccg atacagctaa taagggtgat 780
ttgagtaaca ttccatttgg tactgtcaat ttgttggctt acaataacta ccaagcttaa 840
<210> 5
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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gtagaattaa gatcccgaca catggctcca gccgttgaac aaagatc 47
<210> 6
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<213> Artificial sequence (Artificial Sequence)
<400> 6
ccctaccttc aaattctgga tgagaagact caata 35
<210> 7
<211> 42
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gtgtttacgt tattgatact ggtattgagt cttctcatcc ag 42
<210> 8
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gtctaaggcg aattaattcg cacttaatgg tgatggtgat ggtgagctt 49
<210> 9
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
gcatcttctg ctttggctgc tc 22
<210> 10
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
cgagataggc tgatcaggag caag 24
Claims (9)
1. A proteinase K mutant, characterized in that said mutant is: mutating amino acid 44 of wild type proteinase K, wherein the amino acid sequence of the wild type proteinase K is shown as SEQ ID NO. 1;
the mutants are: alanine at position 44 of wild-type proteinase K was mutated to serine.
2. The proteinase K mutant according to claim 1, wherein the nucleotide sequence encoding the wild-type proteinase K is shown in SEQ ID NO. 2.
3. Proteinase K mutant according to claim 1, characterized in that the amino acid sequence of the mutant is shown in SEQ ID NO. 3.
4. A proteinase K mutant according to claim 3, wherein the nucleotide sequence encoding said mutant is set forth in SEQ ID No. 4.
5. A vector comprising a nucleotide encoding a mutant according to claim 4.
6. A cell comprising the vector of claim 5.
7. A method for the preparation of proteinase K mutant according to any one of claims 1 to 4, comprising the steps of:
step 1, obtaining the nucleotide of the coding mutant shown as SEQ ID NO. 4;
step 2, fusing the nucleotide obtained in the step 1 with an expression vector, constructing a recombinant expression vector, and converting the recombinant expression vector into a host cell Pichia pastoris GS 115;
step 3, inducing host cells containing the recombinant expression vector to express, and separating and purifying to obtain the proteinase K mutant protein;
the proteinase K mutant protein is as follows: alanine 44 of wild-type proteinase K as shown in SEQ ID NO.1 was mutated to serine.
8. The preparation method of claim 7, wherein in step 1, a nucleotide sequence of encoding wild type proteinase K as shown in SEQ ID NO.2 is used as a template, and a nucleotide of encoding mutant as shown in SEQ ID NO.4 is obtained by a site-directed mutagenesis method.
9. A method of increasing proteinase K activity, the method comprising: alanine 44 of wild-type proteinase K as shown in SEQ ID NO.1 was mutated to serine.
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