CN117247923A - Keratinase mutant, engineering bacterium, preparation and application thereof - Google Patents

Abstract

The invention belongs to the field of bioengineering technology, and specifically involves obtaining a keratinase mutant with reduced specific activity of caseinase through site-directed mutation, so as to reduce the degradation effect of the caseinase activity in the keratinase on the casein component in wool, so that the wool fiber has better performance. The mutant is obtained by mutating Q241A, Q241A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, or Q241A/S207A/S213A on the basis of the wild-type keratinase shown in SEQ ID NO.1 . The caseinase activity in the mutant is significantly reduced, which is beneficial to expanding its application in the field of wool textiles.

Description

A keratinase mutant, engineering bacteria and its preparation and application

Technical areas:

The invention belongs to the field of bioengineering technology, and specifically involves obtaining a keratinase mutant with reduced specific activity of caseinase through site-directed mutation, so as to reduce the degradation effect of the caseinase activity in the keratinase on the casein component in wool, so that the wool fiber has better performance.

Background technique:

Keratinase is a special protease. Unlike traditional proteases, keratinase has broad substrate specificity for various insoluble, keratin-rich substrates and can degrade keratin substrates, including feathers and wool. , nails and hair, etc. At the same time, keratinase belongs to the protease class and can also degrade other common protein substrates, such as casein.

Wool is an important raw material in the textile industry. Wool fabric is a soft, warm and comfortable textile. Its structure is curly and divided into scale layer, cortex layer and medulla layer. The scale layer is mainly composed of keratin, which makes wool anti-wear and anti-pollution; the cortex layer is the main component of wool fiber and is composed of proteins such as casein, giving it the advantages of good elasticity and good warmth retention.

Keratinase can degrade keratin and other soluble and insoluble proteins, gradually hydrolyzing them into polypeptides, oligopeptides and free amino acids. In industry, wool fiber has a scale layer arranged from the root of the hair to the tip of the hair. The scale layer not only increases the luster of the wool fabric and improves the resistance to pollution and wear of the wool fabric, but also brings serious felt shrinkage defects to the wool fabric. To improve the effect of enzymatic anti-felting finishing of wool, keratinase is generally used to hydrolyze the scale layer of wool. However, since keratinase can not only degrade keratin, but also has caseinase activity, it can also hydrolyze casein to varying degrees, thereby also It causes damage to the fiber layer of wool, affects the elasticity and breaking strength of wool, and hinders the development and application of keratinase.

Bacillus is the main protease-producing strain and has significant advantages such as short fermentation cycle and rich output. In addition, Bacillus has also made great progress in secreting and expressing foreign proteins. An effective Bacillus expression system has been established. Bacillus has the advantages of protein expression, purification and secretion of active proteins. At the same time, compared with other prokaryotic expression systems , the expression vector of the Bacillus expression system can also use phage as a cloning vector. Nowadays, Bacillus is increasingly used as a genetic engineering expression system.

Therefore, in the present invention, by molecularly modifying the keratinase gene derived from Bacillus licheniformis and performing high-throughput screening using the Bacillus subtilis expression system, a keratinase mutant with reduced caseinase specific activity was obtained.

Contents of the invention:

Since keratinase also has caseinase activity, it will degrade the casein component in wool to varying degrees during the application process, causing varying degrees of damage to wool fibers. In order to reduce the specific activity of caseinase and obtain a keratinase with reduced specific activity of caseinase, it is necessary to To further improve its existing properties.

The purpose of the present invention is to obtain a keratinase mutant with reduced caseinase specific activity. Since the keratinase gene derived from Bacillus licheniformis is a relatively high activity keratinase gene among the keratinases that have been reported so far, it is selected as the starting gene. Modification to reduce caseinase activity. The present invention uses the keratinase gene (bliker) derived from Bacillus licheniformis and the shuttle vector pBSA43 to construct a recombinant expression vector pBSA43-bliker, and expresses it in Bacillus subtilis WB600; through bioinformatics software analysis, its key substrates are determined Combining the regions and key amino acid sites, overlapping PCR technology was used to conduct site-directed mutation of the keratinase gene (bliker) derived from Bacillus licheniformis, and the national standard method (folin phenol method) was used to screen to select caseinase enzymes with reduced specific activity. Keratinase mutants.

The technical route to achieve the purpose of the present invention is summarized as follows:

Site-directed mutagenesis was performed on the keratinase gene (bliker) from B. licheniformis, and the Bacillus subtilis WB600 expression system was used to screen to obtain KER mutants S207A, S213A, T237A, Q241A, and Q241A/S207A with reduced caseinase specific activity. , Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, Q241A/S207A/S213A, and their encoding genes blikerm1, blikerm2, blikerm3, blikerm4, blikerm5, blikerm6, blikerm7, blikerm8, blikerm9, and the screened caseinase KER mutants with reduced specific activity are highly expressed in Bacillus amyloliquefaciens, and KER mutants with lower specific activity of caseinase are obtained through fermentation, extraction and other technologies.

One of the technical solutions provided by the present invention is a keratinase mutant, which is one of the S207A, S213A, T237A, and Q241A mutations that occur on the basis of the wild-type keratinase zymogen region shown in SEQ ID NO.1 at least one acquired;

Further, the keratinase mutant is an S207A mutant, and the amino acid sequence is shown in SEQ ID NO. 3;

Furthermore, the nucleotide sequence of the encoding gene blikerm1 of the S207A mutant is shown in SEQ ID NO.4;

Further, the keratinase mutant is an S213A mutant, and the amino acid sequence is as shown in SEQ ID NO.5;

Furthermore, the nucleotide sequence of the encoding gene blikerm2 of the S213A mutant is shown in SEQ ID NO.6;

Further, the keratinase mutant is a T237A mutant, and the amino acid sequence is shown in SEQ ID NO.7;

Furthermore, the nucleotide sequence of the encoding gene blikerm3 of the T237A mutant is shown in SEQ ID NO.8;

Further, the keratinase mutant is a Q241A mutant, and the amino acid sequence is shown in SEQ ID NO. 9;

Furthermore, the nucleotide sequence of the encoding gene blikerm4 of the Q241A mutant is shown in SEQ ID NO.10;

Further, the keratinase mutant is a Q241A/S207A mutant, and the amino acid sequence is as shown in SEQ ID NO. 11;

Furthermore, the nucleotide sequence of the encoding gene blikerm5 of the Q241A/S207A mutant is shown in SEQ ID NO.12;

Further, the keratinase mutant is a Q241A/S213A mutant, and the amino acid sequence is as shown in SEQ ID NO. 13;

Furthermore, the nucleotide sequence of the encoding gene blikerm6 of the Q241A/S213A mutant is shown in SEQ ID NO.14.

Further, the keratinase mutant is a Q241A/T237A mutant, and the amino acid sequence is as shown in SEQ ID NO.15;

Furthermore, the nucleotide sequence of the encoding gene blikerm7 of the Q241A/T237A mutant is shown in SEQ ID NO.16;

Further, the keratinase mutant is a Q241A/S207A/T237A mutant, and the amino acid sequence is as shown in SEQ ID NO. 17;

Furthermore, the nucleotide sequence of the encoding gene blikerm8 of the Q241A/S207A/T237A mutant is shown in SEQ ID NO. 18;

Further, the keratinase mutant is a Q241A/S207A/S213A mutant, and the amino acid sequence is as shown in SEQ ID NO. 19;

Furthermore, the nucleotide sequence of the encoding gene blikerm9 of the Q241A/S207A/S213A mutant is shown in SEQ ID NO. 20.

The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above mutant encoding gene;

Further, the expression vector used for the recombinant plasmid is pBSA43;

Further, the host cell used by the recombinant strain is Bacillus subtilis or Bacillus amyloliquefaciens;

Furthermore, the host cell is Bacillus subtilis WB600, or the host cell is Bacillus amyloliquefaciens CGMCC No. 11218;

Preferably, the recombinant strain is obtained by connecting the mutant coding gene to the expression vector pBSA43 and then expressing it in the host Bacillus amyloliquefaciens CGMCC No. 11218.

The third technical solution provided by the present invention is the application of the above-mentioned recombinant plasmid or recombinant strain, especially the application in the production of the keratinase mutant described in the first technical solution.

The fourth technical solution provided by the present invention is the application of the keratinase mutant described in the first technical solution, especially its application in hydrolyzing keratin, or its application in anti-felting treatment of wool.

The experimental scheme of the present invention is specifically as follows:

1. Obtaining the KER mutant encoding gene includes the following steps:

(1) Using the wild-type KER encoding gene bliker shown in SEQ ID NO.2 as the starting gene, construct the expression vector pBSA43-bliker and perform site-directed mutagenesis.

(2) The mutated KER encoding gene was transformed into Bacillus subtilis WB600 by constructing a recombinant plasmid, and the specific activity of caseinase was measured using the national standard method.

(3) After screening, KER mutants with reduced caseinase specific activity compared to wild-type keratinase were obtained, and the KER mutant encoding genes blikerm1, blikerm2, blikerm3, blikerm4, blikerm5, blikerm6, blikerm7, blikerm8, and blikerm9 were obtained through sequencing. Plasmids pBSA43-blikerm1, pBSA43-blikerm2, pBSA43-blikerm3, pBSA43-blikerm4, pBSA43-blikerm5, pBSA43-blikerm6, pBSA43-blikerm7, pBSA43-blikerm8, pBSA43-blikerm9 containing genes encoding KER mutants with reduced caseinase specific activity are saved. .

The screened keratinase mutants with reduced casein specific activity are fermented and cultured, and the KER protein is purified.

2. A recombinant strain of Bacillus amyloliquefaciens containing a KER mutant encoding gene and a process for preparing keratinase with reduced casein specific activity, including the following steps:

(1) Connect the KER mutant encoding gene blikerm1-9 to the Bacillus amyloliquefaciens expression plasmid pBSA43 to obtain a new recombinant plasmid pBSA43-blikerm1-9;

(2) The recombinant plasmid pBSA43-blikerm1-9 was transferred into Bacillus amyloliquefaciens CGMCC No. 11218, screened for resistance to Kanamycin (Kan), and verified by enzyme digestion to obtain the recombinant strain, and then the recombinant strain was cultured and fermented. Keratinase is obtained.

The following definitions are used in this invention:

1. Nomenclature of amino acids and DNA nucleic acid sequences

Use the accepted IUPAC nomenclature for amino acid residues, either in single-letter or three-letter code form. DNA nucleic acid sequences adopt the recognized IUPAC nomenclature.

2. Identification of keratinase mutants

"Amino acid replaced at the original amino acid position" is used to represent the mutated amino acid in the KER mutant. For example, Ser207Ala means that the amino acid at position 207 is replaced by Ala from Ser of wild-type KER. The number of the position corresponds to the amino acid sequence number of the wild-type KER zymogen region in SEQ ID NO.1.

In the present invention, bliker in lowercase italics represents the encoding gene of wild-type keratinase KER, blikerm1 in lowercase italics represents the encoding gene of mutant S207A, and blikerm2, blikerm3, blikerm4, blikerm5, blikerm6, blikerm7, blikerm8, and blikerm9 in lowercase italics represent mutations respectively. The coding genes of entities S213A, T237A, Q241A, Q241A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, and Q241A/S207A/S213A. The specific information is as follows.

Beneficial effects:

1. The present invention uses site-directed mutation technology to mutate the KER wild type to obtain mutants S207A, S213A, T237A, Q241A, Q241A/S207A, Q241A/S213A, and Q241A/T237A whose specific activity of caseinase at 60°C is lower than that of the wild type. , Q241A/S207A/T237A, Q241A/S207A/S213A. In the Bacillus subtilis expression system, the specific activity of caseinase of wild-type KER is 949.55U/mg, and the specific activity of caseinase of the mutant is 827.48U/mg and 861.69 respectively. U/mg, 826.70U/mg, 812.76U/mg, 744.11U/mg, 751.97U/mg, 760.97U/mg, 674.88U/mg, 700.41U/mg. The specific activities of keratinase of wild-type KER and each mutant are 750.14U/mg, 703.35U/mg, 775.52U/mg, 752.30U/mg, 772.12U/mg, 796.19U/mg, 789.57U/mg, respectively. 776.19U/mg, 742.36U/mg, 665.38U/mg.

2. Wild-type KER and KER mutants S207A, S213A, T237A, Q241A, Q241A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, Q241A/S207A/S213A were fermented in the Bacillus amyloliquefaciens expression system. The protease activity values are 9685.43U/mL, 8026.53U/mL, 8272.23U/mL, 8184.39U/mL, 7221.25U/mL, 7515.51U/mL, 7369.36U/mL, 7001.04U/mL, 6141.43U/mL, 6583.82U/mL.

3. The present invention uses the Bacillus amyloliquefaciens expression system to achieve efficient expression and preparation of KER mutants with improved enzyme activity.

Picture description:

Figure 1 shows the PCR amplification electrophoresis diagram of the wild-type keratinase zymogen gene.

Among them: M is DNA Marker, 1 is keratinase zymogen gene bliker;

Figure 2 shows the restriction digestion verification diagram of pBSA43-bliker plasmid, in which: M is DNA Marker, 1 is the double restriction digestion diagram of pBSA43-bliker with BamHI and SmaI.

Detailed ways:

The technical content of the present invention will be further described below with reference to the examples. However, the present invention is not limited to these examples, and the protection scope of the present invention cannot be limited by the following examples.

The culture medium used in the embodiment of the present invention is as follows:

LB medium (g/L): yeast extract 5.0, tryptone 10.0, NaCl 10.0, and the rest is water;

Add 2% agar to solid culture medium.

Fermentation medium (g/L): corn flour 64, soybean cake flour 40, add amylase 2.7, Na ₂ HPO ₄ 4, KH ₂ PO ₄ 0.3, and the rest is water; incubate at 90°C for 30 minutes and then sterilize at 121°C for 20 minutes.

In the present invention, the zymogen region sequence of wild-type keratinase KER is shown in SEQ ID NO.1: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDG NGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.

In the present invention, the enzymes of the keratinase S207A mutant are as shown in SEQ ID No.3: MMRKKSFWLGMLTAFMLVFTMAFSASASAQPAKNVEKSGVKDIKQFRIINAKKKKQFRIINAKAKEALDKEAL KEVKNDPDVAYVATHVAHALAQTVPYGIPLIPLIPLIPLIPLIPLIPLIPLIPLIPLIPLIPLIPLIPKGANVAVVAVIQASHPDLNVGGASFVAGEAYNTDGHGHGHGTHVAALDVLGVLNSSGAYSGAYSG IvsgiuWattngMDVINMSLGGSGSGSGSKQAVDNAYARGVVVVAAGNSGNTNTNTNTIGYDSVGAVDSNSNSSNSVGAEVMAPGAGASPTSMASPHVAAALSKH Pnlsasqvrlsstatylgssfyygkglinvegaq.

In the present invention, the enzymes of the keratinase S213A mutant are shown in the SEQ ID No.5: MMRKKSFWLGMLTAFMLVFTMAFSASASAQPAKNVEKSGVKKDIKQFRIINAKKKKQFRIINAKAKEALDKEAL KEVKNDPDVAYVAHAHALAQTVPYGIPLIPLIPLIPLIPLIPLIPLIPLIPLIPLIPLIPLIPKGANVAVAVAVIQASHPDLNVGGASFVAGEAYAGHGHGHGHGTVAALDVLGVLNSSGSGSYSG IvagieWattngMDVINMSLGGSGSGSGSGQAVDNAYARGVVVAAGNSSGNTNTNTNTIGYDSVDSNSNSSNSSNSSNSVGAEVMAPGAGAGAGASPTSMASPHVAAALILSKH Pnlsasqvrlsstatylgssfyygkglinvegaq.

In the present invention, the enzymes of the keratinase T237A mutant are shown in SEQ ID No.7: MMRKKSFWLGMLTAFMLVFTMAFSASASAQPAKNVEKSGVKKDIKQFRIINAKAKKESVDKQFRIINAKAKEALDKEAL KEVKNDPDVAYVAHAHALAQTVPYGIPLIPLIPLIPLIPLIPLIPLIPLIPLIPLIPLIPLIPKGANVAVAVAVIQASHPDLNVGGASFVAGEAYAGHGHGHGHGTVAALDVLGVLNSSGSGSYSG IvsgiuWATTTNGMDVINMSLGGSGSAAMKQAVDNAYARGVVVVVAAGNSSGNTNTNTNTIGYDSVGAVDSNSNSNSSNSVGAGAGAGAGASPTSMASPHVAGAAALSK HPNLSASQVRLSSTATYLGSSFYYGKLINVEGAAQ.

In the present invention, the zymogen region sequence of the keratinase Q241A mutant is shown in SEQ ID NO.9: MMRKKSFLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGE AYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKAAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.

The present invention will be further explained below through specific examples.

Example 1 Obtaining wild-type keratinase gene

1. Use the OMEGA: Bacterial DNA Kit to extract the genomic DNA of Bacillus licheniformis ATCC14580. The extraction steps are as follows:

(1) Use an inoculation loop to inoculate the strain onto an LB solid plate and incubate at 37°C overnight.

(2) Pick a single colony from the culture plate and inoculate it into the liquid test tube culture medium, and culture it overnight at 37°C and 220r/min with shaking.

(3) Take 3 mL-5 mL of bacterial solution and place it in a sterilized EP tube, centrifuge at 12000 r/min for 2 min, and discard the supernatant.

(4) Add 200 μL of sterile water to the EP tube to resuspend the bacterial cells, then add 50 μL of lysozyme, mix by pipetting, and incubate at 37°C for 20 minutes.

(5) Add 100 μL BTL buffer and 20 μL proteinase K to the EP tube, vortex and mix evenly, incubate at 55°C for 40 minutes, and shake and mix every 20 minutes.

(6) Add 5 μL RNase, mix by inverting several times, and place at room temperature for 10 minutes.

(7) Centrifuge at 12000r/min for 2 minutes, remove the undigested part, transfer the supernatant to a new EP tube, add 220μL BDL buffer, and keep in a 65°C water bath for 15min.

(8) Add 220 μL absolute ethanol, pipe and mix evenly.

(9) Transfer the liquid in the EP tube to the recovery column and let it stand for 1 minute. Centrifuge at 12000r/min for 1 minute. Pour the filtrate back into the recovery column. Repeat twice and discard the waste liquid.

(10) Add 500 μL HBC buffer, centrifuge at 12000 r/min for 1 min, and discard the filtrate.

(11) Add 700 μL DNA wash buffer, let it stand for 1 min, centrifuge at 12000 r/min for 1 min, and discard the filtrate.

(12) Add 500 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r/min for 1 min, and discard the filtrate.

(13) Empty for 2 minutes at 12000r/min, discard the waste liquid tube, and put the recovery column into a new EP tube.

(14) Place in a 55°C metal bath to dry for 10 minutes.

(15) Add 50 μL of 55°C sterile water, let stand at room temperature for 5 minutes, centrifuge at 12000 r/min for 2 minutes, discard the recovery column, and the liquid in the EP tube is the genome.

2. Using the extracted genome of Bacillus licheniformis as a template, design a pair of primers upstream and downstream of the ORF box, and introduce restriction enzyme sites BamHI and SmaI respectively. The amplification primers for the keratinase gene bliker of the present invention are as follows:

Upstream primer P1:

5’-CGCGGATCC ATGATGAGGAAAAAAGAGTTTTTGGCT-3’

Downstream primer P2:

5’-TCCCCCGGGTTAGTGATGATGATGATGATGTTGAGCGGCACCTTCGA-3’

P1 and P2 were used as upstream and downstream primers, and the Bacillus licheniformis genome was used as a template for amplification.

The amplification reaction system is:

Upstream primer P1 2.0μL Downstream primer P2 2.0μL DNA template 2.0μL Primer Star Max Enzyme 25μL ddH ₂ O 19μL

The amplification program was: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 57°C for 20 s, extension at 72°C for 6 s, and 30 cycles of reaction; extension at 72°C for 10 min. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis to obtain a 1140 bp band (Figure 1). The PCR product was recovered using a small DNA recovery kit to obtain the wild-type keratinase zymogen region gene bliker (SEQ ID NO. .2). The bliker and pBSA43 plasmids were double digested with the restriction endonucleases BamHI and SmaI respectively. The bliker recovered from the gel cutting was connected to the vector pBSA43 vector to obtain the recombinant plasmid pBSA43-bliker. The restriction enzyme digestion verification is shown in Figure 2, and it was Transformed into Escherichia coli JM109 and Bacillus subtilis WB600, the recombinant Bacillus subtilis strain WB600/pBSA43-bliker was obtained.

Example 2 Construction of a keratinase mutant library to screen keratinase mutants with reduced caseinase specific activity

1. Conduct site-directed mutation based on overlapping PCR technology to construct a new keratinase. Design mutation primers for different mutation sites as follows:

In the first step of the overlapping PCR reaction system, P1 was used as the upstream primer, 207-R, 213-R, 237-R, and 241-R were used as the downstream primers, and the plasmid pBSA43-bliker was used as the template to perform the PCR1 reaction. Obtain the upstream fragment; use P2 as the upstream primer, 207-F, 213-F, 237-F, and 241-F as the downstream primers, and use the plasmid pBSA43-bliker as the template to perform PCR1 reaction to obtain the downstream fragments. Take the S207A mutation as an example:

The reaction system for upstream fragment amplification is:

P1 2μL 207-R 2μL Plasmid pBSA43-bliker 2μL Primer Star Max Enzyme 25μL ddH ₂ O 19μL

The reaction system for downstream fragment amplification is:

P2 2μL 207-F 2μL Plasmid pBSA43-bliker 2μL Primer Star Max Enzyme 25μL ddH ₂ O 19μL

The amplification program was: pre-denaturation at 98°C for 30 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 57°C for 20 s, and extension reaction at 72°C for 6 s; extension at 72°C for 10 min.

2. After cutting the gel and recovering the upstream and downstream fragments, perform PCR2. The reaction system is:

upstream segment 2.0μL downstream fragment 2.0μL Primer Star Max Enzyme 25μL ddH ₂ O 17μL

The amplification program was: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 57°C for 20 s, extension at 72°C for 6 s, and reaction for 5 cycles; extension at 72°C for 10 min.

3. After PCR 2 is completed, add 2 μL each of primers P1 and P2 to the system, and perform PCR 3 amplification program: 98°C pre-denaturation for 30 seconds; 98°C denaturation for 10 seconds, 57°C annealing for 20 seconds, 72°C extension for 6 seconds, and 30 cycles of reaction. ; Extension at 72°C for 10 minutes. The PCR amplification product was subjected to 0.8% agarose gel electrophoresis, and a small DNA recovery kit was used to recover the PCR product, and the keratinase site-directed mutant gene blikerS207A was obtained. In the same way, the corresponding primers were replaced to obtain other keratinase site-directed mutant genes blikerS213A, blikerT237A, and blikerQ241A.

4. Connect the keratinase site-directed mutant genes blikerS207A, blikerS213A, blikerT237A, and blikerQ241A to the expression vector pBSA43 and transform them into E. coli JM109 and extract their plasmids to obtain recombinant plasmids pBSA43-blikerS207A, pBSA43-blikerS213A, pBSA43-blikerT237A, and pBSA4 3- blikerQ241A.

The recombinant plasmids pBSA43-blikerS207A, pBSA43-blikerS213A, pBSA43-blikerT237A, and pBSA43-blikerQ241A were then transformed into Bacillus subtilis WB600 to obtain recombinant strains WB600/pBSA43-blikerS207A, WB600/pBSA43-blikerS213A, and WB60. 0/pBSA43-blikerT237A, WB600/ pBSA43-blikerQ241A. Activate the subtilis-transformed transformant onto a new zoned Kan plate, culture it upside down at 37°C for 12 hours, and then screen the mutant strains. The steps are as follows:

(1) Under sterile conditions, pick a single colony of the mutant and a single colony of the wild type (i.e. WB600/pBSA43-bliker) and inoculate them into a 5 mL liquid LB test tube containing Kan resistance, and culture overnight at 37°C with shaking at 220r/min. .

(2) Take 1 mL of bacterial liquid from the test tube, add it to 50 mL of liquid LB culture medium containing Kan resistance, and culture with shaking at 37°C and 220 r/min for 48 hours.

(3) After the culture is completed, take out the Erlenmeyer flask and measure the bacterial concentration of the bacterial solution at OD600.

(4) Collect the bacterial solution into a 50mL centrifuge tube, place it in a centrifuge, and centrifuge at 8000r/min for 10min. The supernatant will be used as enzyme solution for enzyme activity measurement.

5. Determine the specific activity of casein and keratinase using the national standard method (follin phenol method):

Keratinase hydrolyzes casein or keratin under certain temperature and pH conditions (if not otherwise specified, the temperature of the present invention is 60°C and pH is 10) to produce amino acids containing phenol groups, which are reduced with Folin's phenol reagent to produce tungsten blue , use a UV spectrophotometer to measure the absorbance value of the solution at 680nm. Enzyme activity is proportional to absorbance, and the specific activities of casein and keratinase can be calculated. The measurement method is as follows:

Add 1mL enzyme solution to the blank group, incubate at 60°C for 2 min, add 2 mL of trichloroacetic acid, react at 60°C for 10 min, add 1 mL of casein or keratin (10g/L) solution, take it out and let it stand for 10 min, centrifuge at 12000r/min for 2 min, and take Add 2.5 mL Na ₂ CO ₃ to 0.5 mL of the supernatant, add 0.5 mL of folinol reagent, develop color at 60°C for 20 min, and measure the absorbance of the solution with a UV spectrophotometer at 680 nm using a 10 mm cuvette.

Add 1mL of enzyme solution to the sample group, incubate at 60°C for 2 minutes, add 1mL of casein or keratin (10g/L) solution, react at 60°C for 10min, add 2mL of trichloroacetic acid, take it out and let it stand for 10min, centrifuge at 12000r/min for 2min, and take Add 2.5 mL Na ₂ CO ₃ to 0.5 mL of the supernatant, add 0.5 mL of folinol reagent, develop color at 60°C for 20 min, and measure the absorbance of the solution with a UV spectrophotometer at 680 nm using a 10 mm cuvette.

Subtract the OD value of the blank group from the OD value measured in the sample group to obtain ΔOD, and then substitute ΔOD into the following formula to calculate the corresponding enzyme activity:

(N is the dilution factor of the sample)

At the same time, the mutant strain plasmids pBSA43-blikerS207A, pBSA43-blikerS213A, pBSA43-blikerT237A, and pBSA43-blikerQ241A were sent to Genewise for sequencing. After confirming that each mutation site was correct, the 207th amino acid Ser was mutated into Ala keratinase. The gene of the mutant is named blikerm1, the gene of the keratinase mutant in which amino acid Ser at position 213 is mutated to Ala is named blikerm2, the gene of the keratinase mutant in which amino acid Thr at position 237 is mutated to Ala is named blikerm3, and the gene of the keratinase mutant in which amino acid Thr at position 237 is mutated to Ala is named blikerm3. The gene of the keratinase mutant with Gln mutated to Ala was named blikerm4.

After screening, the mutant Q241A with the lowest caseinase specific activity at 60°C and lower than the wild type was obtained. The mutant Q241A was further combined with S207A, S213A, and T237A to obtain mutants Q241A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, Q241A/S207A/S213A and their encoding genes blikerm5, blikerm6, and blikerm7. , blikerm8, blikerm9. The specific combination mutation process is as follows:

Based on the coding gene of mutant Q241A, a combination of mutation primers was designed, with P1 as the upstream primer, Q241A/S207A-R, Q241A/S213A-R, and Q241A/T237A-R as the downstream primers respectively, P2 as the upstream primer, and Q241A/S207A-F, Q241A/S213A-F, and Q241A/T237A-F were used as downstream primers respectively, and plasmid pBSA43-blikerQ241A was used as the template to perform PCR1 amplification reaction to obtain upstream fragments and downstream fragments respectively. After cutting the gel to recover the upstream and downstream fragments, perform PCR 2. After PCR 2, add primers P1 and P2 to the system and perform PCR 3 to obtain Q241A/S207A, Q241A/S213A, and Q241A/T237A encoding genes blikerm5, blikerm6, and blikerm7. The specific system For conditions and conditions, refer to Steps 1, 2, and 3 of Implementation Example 2.

In the same way, based on the mutant Q241A/S207A, the combined mutation primers Q241A/S207A/T237A-F and Q241A/S207A/T237A-R were designed to amplify the Q241A/S207A/T237 encoding gene blikerm8; the combined mutation primer Q241A/S207A was designed. /S213A-F and Q241A/S207A/S213A-R were amplified to obtain the Q241A/S207A/S213A encoding gene blikerm9.

6. Use the same method as steps 4 and 5 in Example 2 to measure the caseinase and keratinase activities of each mutant. Finally, calculate the caseinase and keratinase specific activities of wild-type KER and each mutant at 60°C, as follows shown in the table.

Keratinase Caseinase specific activity (U/mg) Keratinase specific activity (U/mg) WT 949.55 750.14 S207A 827.48 703.35 S213A 861.69 775.52 T237A 826.70 752.30 Q241A 812.76 772.12 Q241A/S207A 744.11 796.19 Q241A/S213A 751.97 789.57 Q241A/T237A 760.97 776.19 Q241A/S207A/T237A 674.88 742.36 Q241A/S207A/S213A 700.41 665.38

Example 3 Expression and preparation of keratinase mutants in recombinant strains of Bacillus amyloliquefaciens

The KER wild-type encoding gene bliker, and the mutant S207A, S213A, T237A, Q241A, Q241A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, Q241A/S207A/S213A encoding gene blikerm1-9 were compared with the solution respectively. The Bacillus amyloides expression plasmid pBSA43 was connected to obtain new recombinant plasmids pBSA43-bliker, pBSA43-blikerm1, pBSA43-blikerm2, pBSA43-blikerm3...pBSA43-blikerm9;

The recombinant plasmids pBSA43-blikerm1...9 were transferred into Bacillus amyloliquefaciens CGMCCNo.11218, and the wild-type recombinant strain CGMCCNo.11218/pBSA43-bliker and the mutation were obtained through Kana resistance screening and enzyme digestion verification. Recombinant strains CGMCC No.11218/pBSA43-blikerm1, CGMCC No.11218/pBSA43-blikerm2, CGMCC No.11218/pBSA43-blikerm3...CGMCC No.11218/pBSA43-blikerm9.

The Bacillus amyloliquefaciens mutant recombinant strain CGMCC No.11218/pBSA43-blikerm1...9 and the wild-type recombinant strain CGMCC No.11218/pBSA43-bliker were respectively inoculated into 5 mL of fermentation medium (containing kanamycin, 50 μg/mL ), culture overnight at 37°C, 220r/min, transfer to 50mL fresh fermentation medium (containing kanamycin, 50μg/mL) according to 2% inoculation volume, and continue to culture at 37°C, 220r/min for 48h.

Fermentation medium (g/L): corn flour 64, soybean cake flour 40, add amylase 2.7, Na ₂ HPO ₄ 4, KH ₂ PO ₄ 0.3, and the rest is water; incubate at 90°C for 30 minutes and then sterilize at 121°C for 20 minutes.

The fermentation broth was centrifuged to take the supernatant to measure the enzyme activity, and the national standard method in Example 2 was used to measure the caseinase activity obtained by fermentation of Bacillus amyloliquefaciens. In Bacillus amyloliquefaciens, the caseinase activity of wild-type KER is 9685.43U/mL, and the mutants S207A, S213A, T237A, Q241A, Q241A/S207A, Q241A/S213A, Q241A/T237A, Q241A/S207A/T237A, Q241A/S207A The enzyme activities of /S213A are 8026.53U/mL, 8272.23U/mL, 8184.39U/mL, 7221.25U/mL, 7515.51U/mL, 7369.36U/mL, 7001.04U/mL, 6141.43U/mL, 6583.82U/mL .

Preparation of pure protease enzyme powder: Centrifuge the supernatant obtained from the fermentation broth prepared above, first salt out the impurity proteins with ammonium sulfate with a saturation of 25%, and then increase the saturation to 65% to precipitate the target protein. After dissolution, dialyze to remove salt, then dissolve the active component obtained after salting out and desalting with 0.02mol/L Tris-HCl (pH 7.0) buffer, and load it onto a cellulose ion exchange chromatography column using the same buffer. First, elute the unadsorbed protein, and then perform gradient elution with 0.02 mol/L Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0 to 1 mol/L) to collect the target protein. The active components obtained by ion exchange were first equilibrated with 0.02mol/L Tris-HCl (pH 7.0) buffer containing 0.15mol/L NaCl, and then loaded onto a sephadex g25 gel chromatography column with 0.5mL of the same buffer. /min to obtain purified enzyme liquid, which is then freeze-dried to obtain pure keratinase enzyme powder. The prepared keratinase mutant enzyme powder can be used in tanning, food, feed and other fields.

Example 4 Application of keratinase

1. Determination of amino nitrogen content in casein hydrolyzate hydrolyzed by keratinase

Weigh 5g of casein into a beaker and use NaOH solution to help dissolve it. After the casein is completely dissolved, adjust the volume to 100 mL. Take 5 mL of casein solution into two beakers, and put the beakers into a 50°C constant temperature water bath to preheat. Then add 0.1g of wild-type keratinase and mutant Q241A/S207A/T237A to the two beakers. Carry out enzymatic hydrolysis at 50°C for 150 minutes. Stir continuously during the enzymatic hydrolysis. After the reaction, heat the enzymatic hydrolysis solution at 95°C for 10 minutes to inactivate the enzyme, cool to room temperature, and centrifuge at 10,000 r/min for 15 minutes. The amino nitrogen content was determined using a double indicator formaldehyde titration method to determine the remaining casein content in the hydrolyzate. The results showed that the remaining casein content after treatment of casein by wild-type keratinase was 0.96g, and the remaining casein content after treatment by keratinase mutant Q241A/S207A/T237A was 2.28g. Compared with wild-type keratinase, the remaining casein content after treatment of casein by mutant Q241A/S207A/T237A increased by 137.50%, proving that mutant Q241A/S207A/T237A hydrolyzes casein to a lower extent and retains more of it. Casein component.

2. Analysis of mechanical properties of wool

Weigh equal weights of wool (2g), place them in 10mL Gly-NaOH buffer, add 0.1g of wild-type keratinase and mutant Q241A/S207A/T237A, and react in a 50°C water bath shaker for 1 hour. After reaching the reaction time, dry the wool to a constant mass in an oven at 105°C, adjust the humidity for 24 hours in a standard environment with a relative humidity of 65% and a temperature of 20°C, cut it into a test sample with a length of 250mm, and use an INSTRON5590 universal material testing machine. The specimens were tested for elongation at break. Test parameter settings: Stretching at a constant speed, the clamping length of the test fabric test sample is 200mm, and the stretching speed is 200mm/min. Each group of fabrics was tested for 30 groups, and the average value of the test elongation at break was obtained after eliminating abnormal data during the test process.

The results showed that the elongation at break of wool samples treated with wild-type keratinase was 12.86%, and the elongation at break of wool samples treated with mutant Q241A/S207A/T237A was 22.74%, with the elongation at break increased by 76.83%. Since the wool samples treated with the mutant Q241A/S207A/T237A retain more casein components, the elasticity of the wool fiber increases under the same elongation conditions, thereby increasing the elongation at break, thus allowing the wool fabric to maintain better mechanical properties.

Although the present invention has been disclosed above in terms of preferred embodiments, they are not intended to limit the present invention. Any person skilled in the art can make various forms and modifications to these embodiments without departing from the spirit and principles of the present invention. Changes, modifications, substitutions and variations of the details, the scope of the invention is defined by the claims and their equivalents.

Claims (10)

1. A keratinase mutant, characterized in that the mutant produces Q241A, Q241A/S207A, Q241A/S213A, Q241A/T237A, Q241A/ on the basis of the wild-type keratinase shown in SEQ ID NO.1 Obtained by S207A/T237A, or Q241A/S207A/S213A mutation. 2. A keratinase mutant as claimed in claim 2, characterized in that, The amino acid sequence of the Q241A mutant is shown in SEQ ID NO.9; The amino acid sequence of the Q241A/S207A mutant is shown in SEQ ID NO. 11; The amino acid sequence of the Q241A/S213A mutant is shown in SEQ ID NO. 13; The amino acid sequence of the Q241A/T237A mutant is shown in SEQ ID NO. 15; The amino acid sequence of the Q241A/S207A/T237A mutant is shown in SEQ ID NO. 17; The amino acid sequence of the Q241A/S207A/S213A mutant is shown in SEQ ID NO. 19. 3. The coding gene for the keratinase mutant according to claim 1. 4. The coding gene according to claim 3, characterized in that, The nucleotide sequence of the encoding gene of the Q241A mutant is shown in SEQ ID NO.10; The nucleotide sequence of the coding gene for the Q241A/S207A mutant is shown in SEQ ID NO. 12; The nucleotide sequence of the coding gene for the Q241A/S213A mutant is shown in SEQ ID NO. 14; The nucleotide sequence of the coding gene for the Q241A/T237A mutant is shown in SEQ ID NO. 16; The nucleotide sequence of the coding gene for the Q241A/S207A/T237A mutant is shown in SEQ ID NO. 18; The nucleotide sequence of the encoding gene for the Q241A/S207A/S213A mutant is shown in SEQ ID NO. 20. 5. A recombinant plasmid or recombinant strain comprising the encoding gene of claim 3. 6. The recombinant plasmid or recombinant strain according to claim 5, characterized in that the expression vector used is pBSA43, the host cell is Bacillus subtilis WB600, or the host cell is Bacillus amyloliquefaciens CGMCC No. 11218. 7. Use of the recombinant plasmid or recombinant strain of claim 5 in producing the keratinase mutant of claim 1. 8. Use of the keratinase mutant according to claim 1. 9. The application according to claim 8, characterized in that it is used in hydrolyzing keratin. 10. The application as claimed in claim 8, characterized in that it is used in wool anti-felting treatment.