A keratinase mutant, an engineered bacterium and preparation and application thereof

Abstract

The application belongs to the technical field of bioengineering and specifically relates to a keratinase mutant with reduced caseinase specific activity obtained through site-directed mutation, so as to reduce the degradation of the casein component in wool by the caseinase activity in the keratinase, and make the wool fiber have better performance. The mutant is obtained on the basis of the wild-type keratinase shown in SEQ ID NO. 1 through Q241A, Q241A / S207A, Q241A / S213A, Q241A / T237A, Q241A / S207A / T237A or Q241A / S207A / S213A mutation. The caseinase activity in the mutant is significantly reduced, which is conducive to expanding the application of the mutant in the wool textile field.

Description

Technical fields:

[0001] This invention belongs to the field of bioengineering technology, specifically involving obtaining keratinase mutants with reduced specific activity of caseinase through site-directed mutagenesis, so as to reduce the degradation effect of casein activity in keratinase on casein components in wool, thereby giving wool fibers better performance. Background technology:

[0002] Keratinase is a special type of protease. Unlike traditional proteases, keratinase exhibits broad substrate specificity for a wide range of insoluble, keratin-rich substrates, and can degrade keratin substrates, including feathers, wool, nails, and hair. At the same time, keratinase, belonging to the protease class, can also degrade other common protein substrates, such as casein.

[0003] Wool is an important raw material for the textile industry, and wool fabric is a soft, warm, and comfortable textile. Its structure is curly and consists of three layers: the cuticle, the cortex, and the medulla. The cuticle is mainly composed of keratin, which gives wool its abrasion resistance and stain resistance. The cortex is the main component of wool fibers and is composed of proteins such as casein, giving it advantages such as good elasticity and warmth retention.

[0004] Keratinase can degrade keratin and other soluble and insoluble proteins, gradually hydrolyzing them into polypeptides, oligopeptides, and free amino acids. In industry, wool fibers have a scale layer arranged from the hair root to the hair tip. While the scale layer increases the luster of wool fabric and improves its resistance to staining and abrasion, it also causes severe felting defects. To improve the effect of enzymatic anti-felt finishing of wool, keratinase is generally used to hydrolyze the wool scale layer. However, because keratinase can not only degrade keratin but also has casein activity, it can also hydrolyze casein to varying degrees, thereby damaging the wool fiber layer and affecting the elasticity and breaking strength of the wool, thus hindering the development and application of keratinase.

[0005] Bacillus is a major protease-producing strain with significant advantages such as short fermentation cycles and abundant yield. Furthermore, Bacillus has made significant progress in the secretory expression of exogenous proteins, establishing efficient Bacillus expression systems. Bacillus possesses advantages in protein expression, purification, and the secretion of active proteins. Moreover, compared to other prokaryotic expression systems, Bacillus expression systems can utilize bacteriophages as cloning vectors. Today, Bacillus is increasingly widely used as a gene engineering expression system.

[0006] Therefore, in this invention, by molecularly modifying the keratinase gene derived from Bacillus licheniformis and using the Bacillus subtilis expression system for high-throughput screening, a keratinase mutant with reduced casein specific activity was obtained. Summary of the Invention:

[0007] Since keratinase also has casein activity, it will degrade the casein component in wool to varying degrees during application, causing varying degrees of damage to wool fibers. In order to reduce the specific activity of caseinase and obtain keratinase with reduced specific activity, its existing properties need to be further improved.

[0008] The purpose of this invention is to obtain keratinase mutants with reduced caseinase specific activity. Since the keratinase gene from *Bacillus licheniformis* is among the keratinase genes with relatively high activity reported to date, it was chosen as the starting gene for modification to reduce caseinase activity. This invention constructs a recombinant expression vector pBSA43-bliker using the keratinase gene (bliker) from *Bacillus licheniformis* and the shuttle vector pBSA43, and expresses it in *Bacillus subtilis* WB600. Bioinformatics software analysis is used to determine its key substrate-binding regions and key amino acid sites. Site-directed mutagenesis of the keratinase gene (bliker) from *Bacillus licheniformis* is performed using overlap PCR, and the mutants with reduced caseinase specific activity are selected using the national standard method (Folin-Ciocalteu method).

[0009] The technical approach to achieving the objective of this invention is summarized as follows:

[0010] Site-directed mutagenesis was performed on the keratinase gene (bliker) from Bacillus licheniformis. Using the Bacillus subtilis WB600 expression system, KER mutants with reduced caseinase specific activity, namely S207A, S213A, T237A, Q241A, Q241A / S207A, Q241A / S213A, Q241A / T237A, Q241A / S207A / T237A, and Q241A / S207A / S213A, along with their encoding genes blikerm1, blikerm2, blikerm3, blikerm4, blikerm5, blikerm6, blikerm7, blikerm8, and blikerm9, were obtained. These KER mutants with reduced caseinase specific activity were then efficiently expressed in Bacillus amyloliquefaciens, and further processed through fermentation and extraction to obtain KER mutants with even lower caseinase specific activity.

[0011] One of the technical solutions provided by the present invention is a keratinase mutant, which is obtained by at least one of the S207A, S213A, T237A, and Q241A mutations occurring on the basis of the wild-type keratinase progenitor region shown in SEQ ID NO.1;

[0012] Furthermore, the keratinase mutant is the S207A mutant, and its amino acid sequence is shown in SEQ ID NO.3;

[0013] Furthermore, the nucleotide sequence of the gene blikerm1 encoding the S207A mutant is shown in SEQ ID NO.4;

[0014] Furthermore, the keratinase mutant is the S213A mutant, and its amino acid sequence is shown in SEQ ID NO. 5;

[0015] Furthermore, the nucleotide sequence of the gene encoding blikerm2 of the S213A mutant is shown in SEQ ID NO.6;

[0016] Furthermore, the keratinase mutant is a T237A mutant, and its amino acid sequence is shown in SEQ ID NO.7;

[0017] Furthermore, the nucleotide sequence of the gene encoding blikerm3 of the T237A mutant is shown in SEQ ID NO.8;

[0018] Furthermore, the keratinase mutant is the Q241A mutant, and its amino acid sequence is shown in SEQ ID NO.9;

[0019] Furthermore, the nucleotide sequence of the gene encoding blikerm4 of the Q241A mutant is shown in SEQ ID NO.10;

[0020] Furthermore, the keratinase mutant is a Q241A / S207A mutant, with the amino acid sequence shown in SEQ ID NO. 11;

[0021] Furthermore, the nucleotide sequence of the gene blikerm5 encoding the Q241A / S207A mutant is shown in SEQ ID NO. 12;

[0022] Furthermore, the keratinase mutant is a Q241A / S213A mutant, with the amino acid sequence shown in SEQ ID NO. 13;

[0023] Furthermore, the nucleotide sequence of the gene blikerm6 encoding the Q241A / S213A mutant is shown in SEQ ID NO. 14.

[0024] Furthermore, the keratinase mutant is the Q241A / T237A mutant, and its amino acid sequence is shown in SEQ ID NO. 15;

[0025] Furthermore, the nucleotide sequence of the gene blikerm7 encoding the Q241A / T237A mutant is shown in SEQ ID NO. 16;

[0026] Furthermore, the keratinase mutant is a Q241A / S207A / T237A mutant, with the amino acid sequence shown in SEQ ID NO.17;

[0027] Furthermore, the nucleotide sequence of the gene blikerm8 encoding the Q241A / S207A / T237A mutant is shown in SEQ ID NO.18;

[0028] Furthermore, the keratinase mutant is a Q241A / S207A / S213A mutant, with the amino acid sequence shown in SEQ ID NO.19;

[0029] Furthermore, the coding gene blikerm9 of the Q241A / S207A / S213A mutant has the nucleotide sequence shown in SEQ ID NO.20.

[0030] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above-mentioned mutant encoding gene;

[0031] Furthermore, the recombinant plasmid uses pBSA43 as the expression vector;

[0032] Furthermore, the host cell used for the recombinant strain is Bacillus subtilis or Bacillus amyloliquefaciens;

[0033] Furthermore, the host cell is Bacillus subtilis WB600, or the host cell is Bacillus amyloliquefaciens CGMCC No.11218;

[0034] Preferably, the recombinant strain is obtained by ligating the mutant coding gene to the expression vector pBSA43 and then expressing it in the host Bacillus amyloliquefaciens CGMCC No.11218.

[0035] The third technical solution provided by this invention is the application of the above-mentioned recombinant plasmid or recombinant strain, especially its application in the production of the keratinase mutant described in technical solution one.

[0036] The fourth technical solution provided by the present invention is the application of the keratinase mutant described in technical solution one, particularly its application in hydrolyzed keratin, or its application in wool anti-felting treatment.

[0037] The experimental scheme of this invention is as follows:

[0038] 1. Obtaining the gene encoding the KER mutant involves the following steps:

[0039] (1) Using the wild-type KER encoding gene bliker shown in SEQ ID NO.2 as the starting gene, the expression vector pBSA43-bliker was constructed and site-directed mutagenesis was performed.

[0040] (2) The mutated KER encoding gene was transformed into Bacillus subtilis WB600 by constructing a recombinant plasmid, and the specific activity of casein was determined by the national standard method.

[0041] (3) KER mutants with reduced casein activity relative to wild-type keratinase were obtained through screening. The KER mutant encoding genes blikerm1, blikerm2, blikerm3, blikerm4, blikerm5, blikerm6, blikerm7, blikerm8, and blikerm9 were obtained by sequencing. The plasmids pBSA43-blikerm1, pBSA43-blikerm2, pBSA43-blikerm3, pBSA43-blikerm4, pBSA43-blikerm5, pBSA43-blikerm6, pBSA43-blikerm7, pBSA43-blikerm8, and pBSA43-blikerm9 containing the KER mutant encoding genes with reduced casein activity were preserved.

[0042] The keratinase mutant with reduced casein specific activity obtained by screening was fermented and cultured to purify the KER protein.

[0043] 2. The process of preparing keratinase with reduced casein specific activity from recombinant strains of Bacillus amyloliquefaciens containing the KER mutant encoding gene and the process of preparing keratinase with reduced casein specific activity from these strains include the following steps:

[0044] (1) The KER mutant encoding gene blikerm1-9 was ligated with the Bacillus amyloliquefaciens expression plasmid pBSA43 to obtain a new recombinant plasmid pBSA43-blikerm1-9.

[0045] (2) The recombinant plasmid pBSA43-blikerm1-9 was transformed into Bacillus amyloliquefaciens CGMCC No.11218. After screening for kanamycin (Kan) resistance and enzyme digestion verification, the recombinant strain was obtained. The recombinant strain was then cultured and fermented to obtain keratinase.

[0046] In this invention, the following definitions are used:

[0047] 1. Nomenclature of amino acids and DNA nucleic acid sequences

[0048] The IUPAC nomenclature, a recognized system for naming amino acid residues, is used, employing single-letter or three-letter codes. DNA nucleic acid sequences are named using the IUPAC nomenclature.

[0049] 2. Identification of keratinase mutants

[0050] The term "amino acid replaced at the original amino acid position" is used to represent the mutated amino acid in the KER mutant. For example, Ser207Ala indicates that the amino acid at position 207 is replaced by Ala from the wild-type KER, and the position number corresponds to the amino acid sequence number of the wild-type KER proenzyme region in SEQ ID NO. 1.

[0051] In this invention, lowercase italic bliker represents the coding gene for wild-type keratinase KER, lowercase italic blikerm1 represents the coding gene for mutant S207A, and lowercase italic blikerm2, blikerm3, blikerm4, blikerm5, blikerm6, blikerm7, blikerm8, and blikerm9 represent the coding genes for mutants S213A, T237A, Q241A, Q241A / S207A, Q241A / S213A, Q241A / T237A, Q241A / S207A / T237A, and Q241A / S207A / S213A, respectively. Specific information is shown in the table below.

[0052]

[0053] Beneficial effects:

[0054] 1. This invention utilizes site-directed mutagenesis to mutate the wild-type KER enzyme, obtaining mutants S207A, S213A, T237A, Q241A, Q241A / S207A, Q241A / S213A, Q241A / T237A, Q241A / S207A / T237A, and Q241A / S207A / S213A with reduced casein specific activity at 60°C compared to the wild-type. Within the Bacillus expression system, the specific activity of caseinase in wild-type KER was 949.55 U / mg, while the specific activities of caseinase in mutants were 827.48 U / mg, 861.69 U / mg, 826.70 U / mg, 812.76 U / mg, 744.11 U / mg, 751.97 U / mg, 760.97 U / mg, 674.88 U / mg, and 700.41 U / mg, respectively. The specific activities of keratinase in wild-type KER and each mutant were 750.14 U / mg, 703.35 U / mg, 775.52 U / mg, 752.30 U / mg, 772.12 U / mg, 796.19 U / mg, 789.57 U / mg, 776.19 U / mg, 742.36 U / mg, and 665.38 U / mg, respectively.

[0055] 2. The wild-type KER and KER mutants S207A, S213A, T237A, Q241A, Q241A / S207A, Q241A / S213A, Q241A / T237A, Q241A / S207A / T237A, Q241A / S207A / S213A fermented casein enzyme activity values ​​in the Bacillus amyloliquefaciens expression system were 9685.43 U / mL, 8026.53 U / mL, 8272.23 U / mL, 8184.39 U / mL, 7221.25 U / mL, 7515.51 U / mL, 7369.36 U / mL, 7001.04 U / mL, 6141.43 U / mL, and 6583.82 U / mL, respectively.

[0056] 3. This invention uses the Bacillus amyloliquefaciens expression system to achieve efficient expression and preparation of KER mutants with enhanced enzyme activity. Attached image description:

[0057] Figure 1 Electrophoresis diagram of PCR amplification of wild-type keratinaseogen gene

[0058] Where: M is DNA Marker, and 1 is the keratinase progenitor gene bliker;

[0059] Figure 2The image shows the pBSA43-bliker plasmid digestion verification diagram, where M is the DNA Marker and 1 is the pBSA43-bliker double digestion diagram using BamHI and SmaI. Detailed implementation method:

[0060] The technical content of the present invention will be further described below with reference to the embodiments. However, the present invention is not limited to these embodiments, and the scope of protection of the present invention cannot be limited by the following embodiments.

[0061] The culture medium used in the embodiments of this invention is as follows:

[0062] LB medium (g / L): yeast extract 5.0, tryptone 10.0, NaCl 10.0, the remainder being water;

[0063] Add 2% agar to the solid culture medium.

[0064] Fermentation medium (g / L): corn flour 64, soybean meal 40, amylase 2.7, Na2HPO4 4, KH2PO4 0.3, the remainder is water; incubate at 90℃ for 30 min and then sterilize at 121℃ for 20 min.

[0065] In this invention, the zymogen region sequence of wild-type keratinase KER is as shown in SEQ ID NO. Shown in NO.1: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVL GVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.

[0066] In the present invention, the sequence of the zymogen region of the keratinase S207A mutant is as shown in SEQ ID NO. 3: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGAYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.

[0067] In the present invention, the sequence of the zymogen region of the keratinase S213A mutant is as shown in SEQ ID NO. 5: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVAGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.

[0068] In this invention, the zymogen region sequence of the keratinase T237A mutant is as shown in SEQ ID. Shown in NO.7: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVL GVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSAAMKQAVDNAYARGVVVVAAAGNSGSSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.

[0069] In this invention, the zymogen region sequence of the keratinase Q241A mutant is as shown in SEQ ID. Shown in NO.9: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVL GVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKAAVDNAYARGVVVVAAAGNSGSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.

[0070] The present invention will be further explained and illustrated below through specific embodiments.

[0071] Example 1: Obtaining the wild-type keratinase gene

[0072] 1. Genomic DNA was extracted from Bacillus licheniformis (ATCC14580) using the OMEGA Bacterial DNA Kit. The extraction steps are as follows:

[0073] (1) Inoculate the strain onto LB solid plates with an inoculation loop and incubate overnight at 37°C.

[0074] (2) Pick a single colony from the culture plate and inoculate it into a liquid test tube culture medium. Incubate overnight at 37°C with shaking at 220 r / min.

[0075] (3) Take 3-5 mL of bacterial solution and place it in a sterilized EP tube. Centrifuge at 12000 r / min for 2 min and discard the supernatant.

[0076] (4) Add 200 μL of sterile water to the EP tube to resuspend the bacterial cells, then add 50 μL of lysozyme, mix by blowing and aspiration, and keep warm at 37°C for 20 min.

[0077] (5) Add 100 μL of BTL buffer and 20 μL of proteinase K to the EP tube, vortex to mix, incubate at 55°C for 40 min, and vortex to mix every 20 min.

[0078] (6) Add 5 μL of RNase, invert and mix several times, and incubate at room temperature for 10 min.

[0079] (7) Centrifuge at 12000r / min for 2min to remove undigested portion, transfer the supernatant to a new EP tube, add 220μL BDL buffer, and incubate at 65℃ for 15min.

[0080] (8) Add 220 μL of anhydrous ethanol and mix by blowing and sucking.

[0081] (9) Transfer the liquid in the EP tube into the recovery column and let it stand for 1 min. Centrifuge at 12000 r / min for 1 min. Pour the filtrate back into the recovery column and repeat twice. Discard the waste liquid.

[0082] (10) Add 500 μL HBC buffer, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0083] (11) Add 700 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0084] (12) Add 500 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 r / min for 1 min, and discard the filtrate.

[0085] (13) 12000r / min for 2min, discard the waste liquid tube, and put the recovery column into a new EP tube.

[0086] (14) Place in a 55℃ metal bath to dry for 10 minutes.

[0087] (15) Add 50 μL of sterile water at 55℃, let stand at room temperature for 5 min, centrifuge at 12000 r / min for 2 min, discard the recovery column, and the liquid in the EP tube is the genome.

[0088] 2. Using the extracted Bacillus licheniformis genome as a template, a pair of primers were designed upstream and downstream of the ORF frame to introduce restriction enzyme sites BamHI and SmaI, respectively. The amplification primers for the keratinase gene bliker of this invention are as follows:

[0089] Upstream primer P1:

[0090] 5'-CGCGGATCC ATGATGAGGAAAAAAGAGTTTTTGGCT-3'

[0091] Downstream primer P2:

[0092] 5'-TCCCCCGGGTTAGTGATGATGATGATGATGTTGAGCGGCACCTTCGA-3'

[0093] Using P1 and P2 as upstream and downstream primers, amplification was performed using the Bacillus licheniformis genome as a template.

[0094] The amplification reaction system is as follows:

[0095] upstream primer P1 2.0μL Downstream primer P2 2.0μL DNA template 2.0μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0096] The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 57℃ annealing for 20 s, 72℃ extension for 6 s, for 30 cycles; 72℃ extension for 10 min. The PCR amplification products were subjected to 0.8% agarose gel electrophoresis, yielding a 1140 bp band. Figure 1 The PCR product was recovered using a small-volume DNA recovery kit to obtain the wild-type keratinase progenitor gene bliker (SEQ ID NO.2). bliker and the pBSA43 plasmid were double-digested with restriction endonucleases BamHI and SmaI, respectively. The bliker recovered from the gel was ligated into the pBSA43 vector to obtain the recombinant plasmid pBSA43-bliker. Enzyme digestion verification was performed as follows. Figure 2 As shown, it was transformed into Escherichia coli JM109 and Bacillus subtilis WB600 to obtain recombinant Bacillus subtilis strain WB600 / pBSA43-bliker.

[0097] Example 2: Construction of a keratinase mutant library and screening for keratinase mutants with reduced casein specific activity.

[0098] 1. Novel keratinases were constructed by site-directed mutagenesis using overlap PCR technology. Mutation primers were designed for different mutation sites as follows:

[0099]

[0100] In the first step of the overlap PCR reaction system, using P1 as the upstream primer and 207-R, 213-R, 237-R, and 241-R as the downstream primers, with plasmid pBSA43-bliker as the template, PCR1 reaction was performed to obtain the upstream fragment. Similarly, using P2 as the upstream primer and 207-F, 213-F, 237-F, and 241-F as the downstream primers, with plasmid pBSA43-bliker as the template, PCR1 reaction was performed to obtain the downstream fragment. Taking the S207A mutation as an example:

[0101] The reaction system for upstream fragment amplification is as follows:

[0102] P1 2μL 207-R 2μL plasmid pBSA43-bliker 2μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0103] The reaction system for downstream fragment amplification is as follows:

[0104] P2 2μL 207-F 2μL plasmid pBSA43-bliker 2μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 19μL

[0105] The amplification program was as follows: 98℃ pre-denaturation for 30 min; 98℃ denaturation for 10 s, 57℃ annealing for 20 s, 72℃ extension for 6 s, for 30 cycles; 72℃ extension for 10 min.

[0106] 2. After gel extraction and recovery of upstream and downstream fragments, PCR2 was performed. The reaction system was as follows:

[0107] Upstream segment 2.0μL Downstream segments 2.0μL Primer Star Max Enzyme 25μL <![CDATA[ddH2O]]> 17μL

[0108] The amplification program was as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 57℃ annealing for 20s, 72℃ extension for 6s, for 5 cycles; 72℃ extension for 10min.

[0109] 3. After PCR 2, add 2 μL each of primers P1 and P2 to the system. The PCR 3 amplification program is as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 57℃ annealing for 20 s, 72℃ extension for 6 s, 30 cycles; 72℃ extension for 10 min. The PCR products are subjected to 0.8% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit to obtain the site-directed mutant gene blikerS207A of keratinase. Similarly, by replacing the corresponding primers, other site-directed mutant genes blikerS213A, blikerT237A, and blikerQ241A of keratinase are obtained.

[0110] 4. The site-directed mutant genes blikerS207A, blikerS213A, blikerT237A, and blikerQ241A of keratinase were ligated into the expression vector pBSA43 and transformed into Escherichia coli JM109. The plasmids were then extracted to obtain the recombinant plasmids pBSA43-blikerS207A, pBSA43-blikerS213A, pBSA43-blikerT237A, and pBSA43-blikerQ241A.

[0111] 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, WB600 / pBSA43-blikerT237A, and WB600 / pBSA43-blikerQ241A. The transformants from Bacillus subtilis were activated onto a newly streaked Kans plate and incubated upside down at 37°C for 12 hours. The mutant strains were then screened using the following steps:

[0112] (1) Under aseptic conditions, pick single colonies of mutant and wild-type (i.e. WB600 / pBSA43-bliker) and inoculate them into 5 mL of liquid LB tube containing Kan resistance. Incubate overnight at 37°C with shaking at 220 r / min.

[0113] (2) Take 1 mL of bacterial culture from the test tube and add it to 50 mL of liquid LB medium containing Kan resistance. Incubate at 37°C and 220 r / min for 48 h with shaking.

[0114] (3) After the culture is completed, take out the Erlenmeyer flask and measure the bacterial concentration of the bacterial solution at OD600.

[0115] (4) Collect the bacterial culture into a 50 mL centrifuge tube, place it in a centrifuge, centrifuge at 8000 r / min for 10 min, and use the supernatant as the enzyme solution for enzyme activity determination.

[0116] 5. Determination of specific activities of casein and keratinase using the national standard method (Folin-Ciocalteu method):

[0117] Keratinase hydrolyzes casein or keratin under specific temperature and pH conditions (unless otherwise specified, the temperature in this invention is 60℃ and the pH is 10), producing amino acids containing phenolic groups. These amino acids are then reduced with Folin-Ciocalteu reagent to form tungsten blue. The absorbance of the solution is measured at 680 nm using a UV spectrophotometer. Enzyme activity is directly proportional to absorbance, allowing for the calculation of the specific activities of casein and keratinase. The determination method is as follows:

[0118] Add 1 mL of enzyme solution to the blank group, incubate at 60℃ for 2 min, add 2 mL of trichloroacetic acid, react at 60℃ for 10 min, add 1 mL of casein or keratin (10 g / L) solution, remove and let stand for 10 min, centrifuge at 12000 r / min for 2 min, take 0.5 mL of supernatant, add 2.5 mL of Na2CO3, add 0.5 mL of Folin-Ciocalteu reagent, develop color at 60℃ for 20 min, and measure the absorbance of the solution at 680 nm using a 10 mm cuvette with a UV spectrophotometer.

[0119] Add 1 mL of enzyme solution to the sample group, incubate at 60℃ for 2 min, add 1 mL of casein or keratin (10 g / L) solution, react at 60℃ for 10 min, add 2 mL of trichloroacetic acid, remove and let stand for 10 min, centrifuge at 12000 r / min for 2 min, take 0.5 mL of supernatant, add 2.5 mL of Na2CO3, add 0.5 mL of Folin-Ciocalteu reagent, develop color at 60℃ for 20 min, and measure the absorbance of the solution at 680 nm using a 10 mm cuvette with a UV spectrophotometer.

[0120] Subtract the OD value of the blank group from the OD value measured in the sample group to obtain ΔOD. Then, substitute ΔOD into the following formula to calculate the corresponding enzyme activity:

[0121] (N is the dilution factor of the sample)

[0122] Meanwhile, the mutant strain plasmids pBSA43-blikerS207A, pBSA43-blikerS213A, pBSA43-blikerT237A, and pBSA43-blikerQ241A were extracted and sent to Genewiz for sequencing. After confirming that each mutation site was correct, the gene of the keratinase mutant with the 207th amino acid Ser mutated to Ala was named blikerm1, the gene of the keratinase mutant with the 213th amino acid Ser mutated to Ala was named blikerm2, the gene of the keratinase mutant with the 237th amino acid Thr mutated to Ala was named blikerm3, and the gene of the keratinase mutant with the 241st amino acid Gln mutated to Ala was named blikerm4.

[0123] After screening, the mutant Q241A, which exhibited the lowest specific activity of casein at 60℃ and was lower than that of the wild type, was obtained. Further, mutant Q241A was combined with S207A, S213A, and T237A for combined mutations, resulting in mutants Q241A / S207A, Q241A / S213A, Q241A / T237A, Q241A / S207A / T237A, and Q241A / S207A / S213A, along with their encoding genes blikerm5, blikerm6, blikerm7, blikerm8, and blikerm9. The specific combined mutation process is as follows:

[0124] Based on the coding gene of the mutant Q241A, combined mutant primers were designed. P1 was used as the upstream primer, and Q241A / S207A-R, Q241A / S213A-R, and Q241A / T237A-R were used as the downstream primers. P2 was used as the upstream primer, and Q241A / S207A-F, Q241A / S213A-F, and Q241A / T237A-F were used as the downstream primers. Using plasmid pBSA43-blikerQ241A as a template, PCR1 amplification reaction was performed to obtain the upstream and downstream fragments. After gel extraction and recovery of upstream and downstream fragments, PCR2 was performed. After PCR2, primers P1 and P2 were added to the system for PCR3 to obtain the genes encoding Q241A / S207A, Q241A / S213A, and Q241A / T237A, namely blikerm5, blikerm6, and blikerm7. For specific systems and conditions, refer to steps 1, 2, and 3 of Implementation Example 2.

[0125] Similarly, based on the mutant Q241A / S207A, combined mutant primers Q241A / S207A / T237A-F and Q241A / S207A / T237A-R were designed to amplify the Q241A / S207A / T237 encoding gene blikerm8; combined mutant primers Q241A / S207A / S213A-F and Q241A / S207A / S213A-R were designed to amplify the Q241A / S207A / S213A encoding gene blikerm9.

[0126]

[0127] 6. The activities of casein and keratinase in each mutant were determined using the same methods as in steps 4 and 5 of Example 2. The specific activities of casein and keratinase in wild-type KER and each mutant at 60°C were calculated and are shown in the table below.

[0128] keratinase Casein 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

[0129] Example 3: Expression and preparation of keratinase mutant in recombinant Bacillus amyloliquefaciens strain

[0130] The wild-type KER gene encoding bliker, as well as the mutant genes encoding S207A, S213A, T237A, Q241A, Q241A / S207A, Q241A / S213A, Q241A / T237A, Q241A / S207A / T237A, and Q241A / S207A / S213A, blikerm1-9, were ligated with the Bacillus amyloliquefaciens expression plasmid pBSA43 to obtain new recombinant plasmids pBSA43-bliker, pBSA43-blikerm1, pBSA43-blikerm2, pBSA43-blikerm3...pBSA43-blikerm9;

[0131] The recombinant plasmids pBSA43-blikerm1…9 were transformed into Bacillus amyloliquefaciens CGMCCNo.11218, respectively. After screening for kanamycin (Kan) resistance and enzyme digestion verification, wild-type recombinant strain CGMCCNo.11218 / pBSA43-bliker and mutant recombinant strains CGMCC No.11218 / pBSA43-blikerm1, CGMCC No.11218 / pBSA43-blikerm2, CGMCC No.11218 / pBSA43-blikerm3…CGMCC No.11218 / pBSA43-blikerm9 were obtained.

[0132] The Bacillus amyloliquefaciens mutant recombinant strain CGMCC No.11218 / pBSA43-blikerm1…9 and the wild-type recombinant strain CGMCC No.11218 / pBSA43-bliker were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 μg / mL) and cultured overnight at 37 °C and 220 r / min. Then, they were transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 μg / mL) at a 2% inoculation rate and cultured for another 48 h at 37 °C and 220 r / min.

[0133] Fermentation medium (g / L): corn flour 64, soybean meal 40, amylase 2.7, Na2HPO4 4, KH2PO4 0.3, the remainder is water; incubate at 90℃ for 30 min and then sterilize at 121℃ for 20 min.

[0134] The fermentation broth was centrifuged, and the supernatant was collected to determine the enzyme activity. The activity of casein obtained from Bacillus amyloliquefaciens fermentation was determined using the national standard method in Example 2. The casein activity of wild-type KER in Bacillus amyloliquefaciens was 9685.43 U / mL, while the enzyme activities of mutants S207A, S213A, T237A, Q241A, Q241A / S207A, Q241A / S213A, Q241A / T237A, Q241A / S207A / T237A, and Q241A / S207A / S213A were 8026.53 U / mL, 8272.23 U / mL, 8184.39 U / mL, 7221.25 U / mL, 7515.51 U / mL, 7369.36 U / mL, 7001.04 U / mL, 6141.43 U / mL, and 6583.82 U / mL, respectively.

[0135] Preparation of purified protease powder: The supernatant obtained by centrifuging the fermentation broth prepared above was first precipitated with 25% saturated ammonium sulfate to remove impurities, and then the saturation was increased to 65% to precipitate the target protein. After dissolution, the precipitate was dialyzed to remove salt, and the active component obtained after salting-out was dissolved in 0.02 mol / L Tris-HCl (pH 7.0) buffer. The solution was loaded onto a cellulose ion exchange chromatography column, and the unadsorbed protein was eluted with the same buffer. Then, a gradient elution was performed with 0.02 mol / L Tris-HCl (pH 7.0) buffers containing different concentrations of NaCl (0-1 mol / L) to collect the target protein. The active component obtained by ion exchange was first equilibrated with 0.02 mol / L Tris-HCl (pH 7.0) buffer containing 0.15 mol / L NaCl, and then loaded onto a Sephadex G25 gel chromatography column. The solution was eluted with the same buffer at a rate of 0.5 mL / min to obtain purified enzyme solution. After freeze-drying, the purified keratinase powder was obtained. The prepared keratinase mutant enzyme powder can be used in leather making, food, feed and other fields.

[0136] Example 4: Application of Keratinase

[0137] 1. Determination of amino nitrogen content in casein hydrolysate hydrolyzed by keratinase

[0138] Weigh 5g of casein into a beaker and dissolve it with NaOH solution until completely dissolved. Then, bring the volume to 100mL. Take 5mL of the casein solution into two separate beakers and preheat them in a 50℃ water bath. Add 0.1g of wild-type keratinase and mutant Q241A / S207A to each beaker, respectively. Incubate at 50℃ for 150min with constant stirring. After the reaction, heat the hydrolysate at 95℃ for 10min to inactivate the enzyme, cool to room temperature, and centrifuge at 10000r / min for 15min. Determine the amino nitrogen content using a double-indicator formaldehyde titration method to determine the residual casein content in the hydrolysate. The results showed that the residual casein content after treatment with wild-type keratinase was 0.96g, and the residual casein content after treatment with keratinase mutants Q241A / S207A / T237A was 2.28g. Compared to wild-type keratinase, the casein content remaining after treatment with mutant Q241A / S207A / T237A increased by 137.50%, demonstrating that mutant Q241A / S207A / T237A hydrolyzes casein to a lower degree and retains more of its casein components.

[0139] 2. Mechanical property analysis of wool

[0140] Equal weights of wool (2g) were weighed and placed in 10mL of Gly-NaOH buffer. 0.1g of wild-type keratinase and mutant Q241A / S207A / T237A were added separately, and the mixture was reacted in a 50℃ water bath shaker for 1 hour. After the reaction time was reached, the wool was dried to constant weight in an oven at 105℃ and conditioned for 24 hours in a standard environment with 65% relative humidity and 20℃. Test specimens were cut to a length of 250mm and tested for elongation at break using an INSTRON 5590 universal testing machine. Test parameters were set as follows: uniform stretching, test specimen clamping length 200mm, stretching speed 200mm / min. Thirty sets of tests were conducted for each fabric, and the average elongation at break was taken after removing abnormal data.

[0141] The results showed that the breaking elongation of the wool sample treated with wild-type keratinase was 12.86%, while the breaking elongation of the wool sample treated with mutant Q241A / S207A / T237A was 22.74%, representing an increase of 76.83%. Because the wool sample treated with mutant Q241A / S207A / T237A retained more casein, it increased the elasticity of the wool fiber under the same elongation conditions, thus increasing the breaking elongation and maintaining better mechanical properties of the wool fabric.

[0142] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A keratinase mutant, characterized in that, The mutant was obtained by a Q241A / S213A mutation on the wild-type keratinase shown in SEQ ID NO.1; the amino acid sequence of the mutant is shown in SEQ ID NO.

13.

2. The encoding gene of the keratinase mutant of claim 1.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

14.

4. A recombinant plasmid or recombinant strain containing the encoding gene of claim 2.

5. The recombinant plasmid or recombinant bacterial strain as described in claim 4, characterized in that, The expression vector used was pBSA43, and the host cell was Bacillus subtilis WB600, or Bacillus amyloliquefaciens CGMCC No.11218.

6. The use of the recombinant plasmid or recombinant strain of claim 4 in the production of the keratinase mutant of claim 1.

7. The use of the keratinase mutant of claim 1 in hydrolyzed keratin.

8. The application of the keratinase mutant of claim 1 in wool anti-felting treatment.

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