CN113913412A - Proteinase K mutant and preparation method thereof - Google Patents
Proteinase K mutant and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a proteinase K mutant and a preparation method thereof, belonging to the technical field of protein engineering, wherein the mutant is as follows: mutating the 44 th amino acid of wild proteinase K shown as SEQ ID NO.1, specifically, mutating the 44 th alanine into serine. The invention takes the crystal structure of the proteinase K as the basis, and modifies the proteinase K through technologies such as protein engineering, site-directed mutagenesis and the like, thereby obtaining the mutant A44S shown in SEQ ID NO.3 for the first time. Researches find that the specific enzyme activity of the mutant A44S is improved by about 21% compared with that of a wild type, so that the application cost of the proteinase K is reduced, the industrial application value of the enzyme in various industries such as washing, feed and food is improved, and an important clue is provided for the research of a 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 protease derived from Candida albicans (Tritirachium album limber), has extremely high enzymatic activity and a broad substrate spectrum, has high cleavage ability to natural proteins under a broad spectrum of pH conditions, particularly alkaline conditions, is preferentially superior to cleavage of carboxyl-terminal peptide bonds of aliphatic amino acids and aromatic amino acids, and preferentially decomposes ester bonds and peptide bonds adjacent to the C-terminus of hydrophobic amino acids, sulfur-containing amino acids and aromatic amino acids. In 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 used as an important enzyme for degrading protein and extracting RT-qPCR template, and the market demand is increased dramatically. Meanwhile, the enzyme has good resistance to SDS, urea and the like, so that the enzyme shows good application effect in the fields of washing industry, feed industry, sewage treatment, paper making, food and the like.
Gunkel et al achieved heterologous expression of proteinase K in E.coli cells for the first time in 1989 and studied its basic properties. With the advance of the 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 bound2+It has important promotion effect on activity and thermal stability. However, the expression level of the enzyme in Candida albicans is very low, the enzyme is difficult to be applied to actual production, and although the enzyme has high activity under alkaline conditions, can tolerate a certain concentration of detergent and has good application prospect in the washing industry, the activity of the enzyme has a certain difference compared with the activity of the protease for foreign commercial washing. Therefore, how to modify proteinase K to improve the expression level and the enzyme activity is of great significance.
Disclosure of Invention
The invention aims to provide a protease K mutant, which is based on the crystal structure of protease K, improves the specific activity of the protease by modifying the protease molecule through a rational design method, and provides possibility for the application of the protease in the washing industry.
One of the purposes of the invention is to provide a protease K mutant which is: mutating the 44 th amino acid of the wild proteinase K, wherein the amino acid sequence of the wild proteinase K is shown as SEQ ID NO. 1.
Further, the nucleotide sequence for coding the wild-type proteinase K is shown as SEQ ID NO. 2.
Further, the mutant is: alanine at position 44 of wild-type proteinase K was mutated to serine.
Furthermore, the amino acid sequence of the mutant is shown as SEQ ID NO. 3.
Further, the nucleotide sequence for coding the mutant is shown as SEQ ID NO. 4.
The second object of the present invention is to provide a vector comprising the above-mentioned nucleotide encoding the mutant.
It is a further object of the present invention to provide a cell comprising the vector.
The fourth purpose of the invention is to provide a preparation method of the protease K mutant, which comprises the following steps:
and 3, inducing the host cell 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 coding wild type proteinase K shown as SEQ ID NO.2 is used as a template, and the nucleotide of the coding mutant shown as SEQ ID NO.4 is obtained by adopting a site-specific mutagenesis method.
Further, the method of site-directed mutagenesis comprises: taking a nucleotide sequence which is shown as SEQ ID NO.2 and codes a wild type proteinase K as a template, and respectively taking F primer, A44S-R primer, A44S-F primer and R primer as primer pairs to carry out PCR amplification to obtain two sections of PCR products taking a mutation point as two ends; and then, taking the two PCR products as templates, taking the F primer and the R primer as primers to carry out PCR, and connecting the two PCR products 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 as SEQ ID NO. 6; the nucleotide sequence of the A44S-F primer is shown as SEQ ID NO. 7; the nucleotide sequence of the R primer is shown as SEQ ID NO. 8.
The fifth purpose of the invention is to provide a method for improving the activity of proteinase K, which comprises the following steps: alanine at position 44 of wild type proteinase K shown in SEQ ID NO.1 is mutated into 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 the proteinase K on the basis of the crystal structure of the proteinase K, modifies the proteinase K by the technologies of protein engineering, site-directed mutagenesis and the like, and obtains a mutant A44S of mutating alanine at the 44 th position into serine for the first time, wherein the amino acid sequence of the mutant is shown as SEQ ID NO. 3. Researches find that the specific enzyme activity of the mutant A44S is improved by about 21% compared with that of a wild type, so that the application cost of the proteinase K is reduced, the industrial application value of the enzyme in various industries such as washing, feed and food is improved, and an important clue is provided for the research of a catalytic mechanism of the enzyme.
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FIG. 1 shows SDS-PAGE of wild-type proteinase K and its mutant A44S in example 3, 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 described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. Acquisition of A44S mutant
Taking the nucleotide sequence of the coding wild type proteinase K shown in SEQ ID NO.2 as a template, respectively taking F primer, A44S-R primer, A44S-F primer and R primer as primer pairs, and respectively carrying out conventional PCR amplification to obtain two sections of PCR products taking a mutation point as two ends. And performing conventional PCR amplification by using the two PCR products as templates and using F primer and R primer as primers, and connecting the two PCR products 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 (shown in SEQ ID NO. 5); A44S-R primer: ccctaccttcaaattctggatgagaAGActcaata (shown in SEQ ID NO. 6); A44S-F primer: gtgtttacgttattgatactggtattgagTCTtctcatccag (shown in SEQ ID NO. 7); r primer: gtctaaggcgaattaattcgcacttaatggtgatggtgatggtgagctt (shown in SEQ ID NO. 8),
wherein the capital part in A44S-R primer and A44S-F primer is the mutation site.
2. Construction of recombinant expression vectors
The Pichia pastoris expression vector pHBM905BDM is subjected to double digestion by Not I and Cpo I, recovered and mixed with the coding gene fragment of the mutant A44S obtained by PCR amplification, and 0.1U of T5 exonuclease is added to treat the mixture for 3 to 5min on an ice water mixture. Then the strain is transferred into Escherichia coli XL10-Gold competent cells. The transformation liquid was spread on LB plate containing ampicillin (100mg/L), the plasmid was extracted, and the constructed recombinant plasmid was verified by colony PCR and named pHBM905 BDM-A44S. The sequencing work is completed by Shanghai worker.
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 screening of recombinant proteinase K mutant plasmids in Pichia pastoris
(1) Recombinant proteinase K mutant expression vectors prepared in the examplesThe pHBM905BDM-A44S 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 cut by restriction enzyme SalI, and then glue is made for recovery, 10 mu L of linear fragment is taken to be uniformly mixed with 100 mu L of GS115 yeast competent cells, and the mixture is transferred to an electric rotating cup (the gap between two electrodes is 2mm) pre-cooled by ice, and is put into an electric rotating instrument for electric shock, and the electric shock parameters are as follows: 1500V, 25. mu.F, 200. omega.; then, 200 mu L of 1M sorbitol is quickly added into the electric rotating cup, 200 mu L of MD culture medium is added, the mixture is evenly mixed and then is transferred into a 1.5mL EP tube, and the shaking table is activated for 2 hours at the temperature of 28 ℃; the cell suspension was centrifuged to remove the supernatant, added with 80. mu.L of MD medium for resuspension, and spread on MD plates (glucose 20.0g/L, (NH)4)2SO410.0g/L, YNB 13.4g/L, agar 2%, 500 × Biotin 0.02%, 2mL), and inverted in an incubator at 28 ℃ until a single colony appears.
(2) Simultaneously adding the transformed yeast single colony to the BMGY plate and the MD plate, wherein the same single colony is marked consistently; after about 36-48h, the colonies on the BMGY plate were spotted onto 1% casein BMMY plates and the marks were kept consistent; adding 200 mu L of methanol on filter paper every 12h for induction; observing a hydrolysis ring on the casein plate after the induction is carried out for 48-60 hours, wherein a single bacterial colony corresponding to the hydrolysis ring is a proteinase K recombinant bacterial colony; culturing the recombinant colony with hydrolysis loop to OD6000.2-0.3, 500. mu.L of the bacterial liquid and equal volume of 50% glycerol are mixed in a freeze-drying tube and stored in a refrigerator at-80 ℃.
Example 3 Shake flask expression and enzymatic Properties determination of wild-type proteinase K and its mutants in Pichia pastoris
(1) Respectively inoculating wild proteinase K and A44S mutant expression strains in l00mL BMGY medium, and shake-culturing at 28 deg.C to OD600Is 6-10. After centrifugation at 3000rpm for 5min, the supernatant was discarded, and the cells were collected and resuspended in 50mL of BMMY medium. Shaking-culturing at 28 deg.C, adding methanol every 12h to final concentration of 1%, continuously inducing for about 120h, and periodically sampling and detecting. After 120h of induction, the induced bacterial liquid is centrifuged (4000rpm, 5min), the supernatant is filtered through a 0.22 μm filter membrane, and the filtrate is stored in a sterile 50mL centrifuge tube and stored in a refrigerator at 4 ℃.
(2) And (3) protein ultrafiltration liquid changing, namely transferring the filtered protein into a 10kD ultrafiltration tube, centrifuging at 4000rpm and 4 ℃, changing the liquid by using 3 times of volume of PBS buffer solution, and finally storing the concentrated proteinase K at 4 ℃ for later use. The wild-type proteinase K and the A44S mutant were detected by SDS-PAGE gel, and the detection results are shown in FIG. 1, wherein lane 1 is the wild-type proteinase K and lane 2 is the A44S mutant. Meanwhile, a standard curve is drawn by using a Bradford protein concentration determination kit, and the corresponding protein concentration is calculated.
(3) And (3) enzyme activity determination: the enzyme activity is defined as that the enzyme amount for hydrolyzing casein to generate 1 mu g of tyrosine within 1min under certain temperature and pH conditions is one enzyme activity unit and is expressed by U. The determination method mainly refers to the national standard BG/T28715-2012, but the reaction system is reduced. The reaction process is as follows: adding 100 μ L enzyme solution into 1.5mL EP tube, preheating in 55 deg.C water bath for 3min, adding 100 μ L casein solution preheated in the same way, shaking, adding 200 μ L trichloroacetic acid solution in 55 deg.C water bath for 10min, and shaking (adding trichloroacetic acid and casein solution into blank control). The mixture was taken out, cooled to room temperature, and centrifuged at 12000rpm for 10 min. Collecting supernatant 100 μ L, adding sodium carbonate solution 500 μ L, adding diluted Folin reagent (commercialized Folin: ddH)2O is 1: 2)100 μ L, developing at 40 deg.C for 20min, and measuring absorbance at 680nm wavelength with enzyme-labeling instrument.
The results of the wild-type and mutant protein concentration and enzyme activity measurements are shown in Table 1.
TABLE 1 determination of the concentration and the specific enzyme activity of wild-type proteinase K and its mutants
The results of fig. 1 and table 1 show that the expression level of the proteinase K mutant a44S prepared by the invention is slightly reduced compared with that of wild proteinase K, but the enzyme activity is significantly improved, and the specific enzyme activity is improved by about 21%, that is, the mutant a44S is obtained by mutating wild proteinase K, so that the application cost is reduced, the industrial application value of the enzyme in various industries such as washing, feed and food is improved, and an important clue is provided for the catalytic mechanism research of the enzyme.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Sequence listing
<110> university of Hubei
<120> proteinase K mutant and method for producing the same
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 279
<212> PRT
<213> Linburgh Candida albicans (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
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Ser Cys Val Tyr Val Ile Asp Thr Gly Ile Glu Ala Ser His Pro Glu
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Phe Glu Gly Arg Ala Gln Met Val Lys Thr Tyr Tyr Ala Ser Ser Arg
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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
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Asp Asp Asn Gly Ser Gly Gln Tyr Ser Thr Ile Ile Ala Gly Met Asp
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Phe Val Ala Ser Asp His Asn Asn Arg Asn Cys Pro Lys Gly Val Val
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Ala Ser Leu Ser Leu Gly Gly Gly Tyr Ser Ser Ser Val Asn Ser Ala
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Ala Ala Arg Leu Gln Ser Ser Gly Val Met Val Ala Val Ala Ala Gly
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Asn Asn Asn Ala Asp Ala Arg Asn Tyr Ser Pro Ala Ser Glu Pro Ser
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Val Cys Thr Val Gly Ala Thr Asp Arg Tyr Asp Arg Arg Ser Ser Phe
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Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Ala Pro Gly Thr Ser Ile
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Leu Ser Thr Trp Ile Gly Gly Ser Thr Arg Ser Ile Ser Gly Thr Ser
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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
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<210> 2
<211> 840
<212> DNA
<213> Linburgh Candida albicans (Tritirachium album limber)
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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
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<213> Artificial Sequence (Artificial Sequence)
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Ala Ala Gln Thr Asn Ala Pro Trp Gly Leu Ala Arg Ile Ser Ser Thr
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Asn Asn Asn Ala Asp Ala Arg Asn Tyr Ser Pro Ala Ser Glu Pro Ser
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Val Cys Thr Val Gly Ala Thr Asp Arg Tyr Asp Arg Arg Ser Ser Phe
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Ser Asn Tyr Gly Ser Val Leu Asp Ile Phe Ala Pro Gly Thr Ser Ile
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Leu Ser Thr Trp Ile Gly Gly Ser Thr Arg Ser Ile Ser Gly Thr Ser
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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
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Ala Tyr Asn Asn Tyr Gln Ala
275
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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
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gtagaattaa gatcccgaca catggctcca gccgttgaac aaagatc 47
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ccctaccttc aaattctgga tgagaagact caata 35
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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gtctaaggcg aattaattcg cacttaatgg tgatggtgat ggtgagctt 49
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<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gcatcttctg ctttggctgc tc 22
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cgagataggc tgatcaggag caag 24
Claims (10)
1. A proteinase K mutant, wherein the mutant is: mutating the 44 th amino acid of the wild proteinase K, wherein the amino acid sequence of the wild proteinase K is shown as SEQ ID NO. 1.
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. The proteinase K mutant according to claim 1, wherein the mutant is: alanine at position 44 of wild-type proteinase K was mutated to serine.
4. The proteinase K mutant according to claim 3, wherein the amino acid sequence of the mutant is shown in SEQ ID No. 3.
5. The proteinase K mutant according to claim 4, wherein the nucleotide sequence encoding the mutant is shown in SEQ ID No. 4.
6. A vector comprising the nucleotide encoding the mutant as claimed in claim 5.
7. A cell comprising the vector of claim 6.
8. The method for producing proteinase K mutant according to any one of claims 1 to 5, 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 to construct a recombinant expression vector, and converting the recombinant expression vector into a host cell Pichia pastoris GS 115;
and 3, inducing the host cell containing the recombinant expression vector to express, and separating and purifying to obtain the proteinase K mutant protein.
9. The method according to claim 8, wherein the nucleotide sequence of wild-type proteinase K shown in SEQ ID No.2 is used as a template in step 1, and the nucleotide sequence of the coding mutant shown in SEQ ID No.4 is obtained by site-directed mutagenesis.
10. A method of increasing proteinase K activity, comprising: alanine at position 44 of wild type proteinase K shown in SEQ ID NO.1 is mutated into serine.
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Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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