Keratinase mutant and its application

Abstract

The present invention belongs to the field of bioengineering technology and specifically relates to obtaining keratinase mutants with enhanced enzyme activity through site-directed mutagenesis. The present invention utilizes overlapping PCR technology to perform site-directed mutagenesis on a keratinase gene derived from Bacillus licheniformis. Keratinase mutants with enhanced enzyme activity are screened and expressed in Bacillus subtilis and Bacillus amyloliquefaciens systems. The resulting keratinase mutants can be used in keratin hydrolysis, particularly in the feed processing, pharmaceutical, leather, and cosmetics industries, addressing the problem of efficient keratin hydrolysis.

Description

Technical field:

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a keratinase mutant with improved enzyme activity obtained through site-directed mutagenesis. Background technology:

[0002] Keratin is a hard, insoluble protein widely found in nature, primarily in animal hair, scales, feathers, hooves, and horns. Its chemical structure is stable and insoluble in water, dilute acids, and alkalines. Agricultural production generates millions of tons of keratin-rich waste and byproducts annually. These wastes contain high levels of protein, making them valuable for recycling and reuse. However, current methods for keratin degradation employ chemical or physical methods, which hinder waste utilization. Acid-base hydrolysis also produces acidic and alkaline wastewater and steam, which pose health risks and pollute the environment. Enzymatic degradation of keratin, by contrast, offers significant potential for development, characterized by lower costs, milder processing conditions, less loss of protein nutritional value, and higher amino acid utilization.

[0003] Keratinase is a protease with a very broad substrate range, capable of degrading a variety of soluble and insoluble proteins, such as collagen, fibrin, bovine whey protein, hemoglobin, and casein. Most notably, it can specifically degrade natural keratin, particularly those with a high sulfur content, catalyzing its denaturation and hydrolysis into soluble amino acids and peptides. This activity is also mild, offering significant advantages over other traditional proteases. The discovery of keratin has provided an ideal new way to transform keratin waste into valuable resources, but further research is needed to enhance the activity of keratinase and improve its keratin degradation capabilities.

[0004] Directed evolution of enzymes is a rational design of proteins. By artificially creating special evolutionary conditions that mimic natural evolutionary mechanisms, enzyme genes are modified in vitro, and targeted screening is performed to obtain enzymes with desired characteristics. Numerous enzymes have been successfully modified using site-directed mutagenesis, resulting in industrial enzymes with higher activity and better stability than natural enzymes. Some progress has been made in the field of subtilisin. It has been discovered that the steric hindrance and electrostatic distribution of the substrate-binding regions of subtilisin are closely related to its substrate specificity, with the S1 and S4 substrate-binding regions playing a major role. Therefore, research on the modification of these S1 and S4 substrate-binding regions has become a key focus.

[0005] Bacillus subtilis has a more prominent advantage in secreting and expressing keratinase. Bacillus subtilis also has a relatively complete protein folding and secretion mechanism. There is basically no codon preference problem when expressing keratinase genes from Bacillus, which enables keratinase from Bacillus to be overexpressed in the Bacillus subtilis host.

[0006] Therefore, in the present invention, a keratinase gene derived from Bacillus licheniformis is molecularly modified and a Bacillus subtilis expression system is used for high-throughput screening to obtain a keratinase mutant with improved enzyme activity. Summary of the invention:

[0007] In order to increase the enzymatic activity of keratinase and obtain highly active keratinase mutants, its existing properties need to be further improved.

[0008] The present invention aims to obtain keratinase mutants with enhanced enzymatic activity. A keratinase gene derived from Bacillus licheniformis is used as a starting gene for molecular modification. The present invention combines the Bacillus licheniformis keratinase gene (bliker) with the shuttle vector pBSA43 to construct a recombinant expression vector pBSA43-bliker, which is then expressed in Bacillus subtilis WB600. Bioinformatics software analysis is used to determine the key substrate binding region and key amino acid sites. Overlapping PCR is used to perform site-directed mutagenesis on the Bacillus licheniformis keratinase gene (bliker). Screening is performed using the national standard method (Folin-phenol method) to select keratinase mutants with enhanced enzymatic activity.

[0009] One of the technical solutions provided by the present invention is a keratinase mutant, which is obtained by L136A mutation occurring in the zymogen region of the wild-type keratinase shown in SEQ ID NO.1;

[0010] Furthermore, the keratinase mutant is an L136A mutant, and the amino acid sequence is shown in SEQ ID NO.3;

[0011] Furthermore, the nucleotide sequence of the gene blikerm1 encoding the L136A mutant is shown in SEQ ID NO.4.

[0012] The second technical solution provided by the present invention is a recombinant plasmid or recombinant strain containing the above mutant encoding gene;

[0013] Furthermore, the expression vector used in the recombinant plasmid is pBSA43;

[0014] Furthermore, the host cell used by the recombinant strain is Bacillus subtilis or Bacillus amyloliquefaciens;

[0015] Furthermore, the host cell is Bacillus subtilis WB600, or the host cell is Bacillus amyloliquefaciens CGMCC No.11218;

[0016] Preferably, the recombinant strain is obtained by connecting the mutant encoding gene to the expression vector pBSA43 and expressing it in the host Bacillus amyloliquefaciens CGMCC No.11218.

[0017] The third technical solution provided by the present invention is the application of the above-mentioned recombinant plasmid or recombinant strain, especially in the production of L136A mutant.

[0018] The fourth technical solution provided by the present invention is the application of the L136A mutant, particularly in the hydrolysis of keratin, and more particularly in the feed processing industry, the pharmaceutical industry, the leather industry and the cosmetics industry.

[0019] The experimental scheme of the present invention is as follows:

[0020] 1. Obtaining the gene encoding the KER mutant includes the following steps:

[0021] (1) Using the wild-type KER encoding gene bliker shown in SEQ ID NO.2 as the starting gene, an expression vector pBSA43-bliker was constructed and site-directed mutagenesis was performed.

[0022] (2) The mutated coding gene was transferred into Bacillus subtilis WB600 by constructing a recombinant plasmid, and the keratinase activity was determined using the national standard method.

[0023] (3) The KER mutant encoding gene blikerm1 with improved keratinase activity compared to the wild type was obtained through screening, and the plasmid pBSA43-blikerm1 containing the KER mutant encoding gene with improved keratinase activity was preserved.

[0024] The screened keratinase mutant with improved keratinase activity is fermented and cultured, and the KER mutant protein is purified.

[0025] 2. A recombinant strain of Bacillus amyloliquefaciens containing a gene encoding a KER mutant and a process for preparing a keratinase mutant using the same comprises the following steps:

[0026] (1) The KER mutant encoding gene blikerm1 was ligated with the Bacillus amyloliquefaciens expression plasmid pBSA43 to obtain a new recombinant plasmid pBSA43-blikerm1;

[0027] (2) The recombinant plasmid pBSA43-blikerm1 was transformed into Bacillus amyloliquefaciens CGMCC No.11218, and the recombinant strain was obtained by kanamycin (Kan) resistance screening and enzyme digestion verification. The recombinant strain was then cultured and fermented to obtain a keratinase mutant.

[0028] The following definitions are used in the present invention:

[0029] 1. Nomenclature of amino acid and DNA sequences

[0030] The generally accepted IUPAC nomenclature for amino acid residues is used, using either the single-letter or three-letter code. DNA nucleic acid sequences use the generally accepted IUPAC nomenclature.

[0031] 2. Identification of Keratinase Mutants

[0032] The term "amino acid substituted at the original amino acid position" is used to represent the mutated amino acid in the KER mutant. For example, Leu136Ala indicates that the amino acid at position 136 is substituted from Leu in wild-type KER to Ala. The position number corresponds to the amino acid sequence number of the wild-type KER zymogen region in SEQ ID NO.1.

[0033] In the present invention, the lowercase italic "bliker" indicates the gene encoding the wild-type keratinase KER, and the lowercase italic "blikerm1" indicates the gene encoding the mutant L136A. The specific information is shown in the following table.

[0034]

[0035] Beneficial effects:

[0036] 1. The present invention utilizes site-directed mutagenesis technology to mutate the wild-type KER to obtain a mutant L136A having improved keratinase activity at 60°C relative to the wild-type. In a Bacillus subtilis expression system, the specific keratinase activities of the wild-type KER and the mutant L136A were 780.5 U / mg and 1069.4 U / mg, respectively.

[0037] 2. The present invention uses the Bacillus amyloliquefaciens expression system to achieve efficient expression and preparation of KER mutants with improved enzyme activity. Description of the drawings:

[0038] Figure 1 The electrophoresis diagram of the wild-type keratinase zymogen gene amplified by PCR

[0039] Among them: M is DNA Marker, 1 is keratinase zymogen gene bliker;

[0040] Figure 2The figure shows the restriction enzyme digestion verification of pBSA43-bliker plasmid, where M is a DNA marker and 1 is a double enzyme digestion image of pBSA43-bliker by BamHI and SmaI. Specific implementation method:

[0041] The technical content of the present invention will be further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0042] The culture medium used in the embodiments of the present invention is as follows:

[0043] LB medium (g / L): yeast extract 5.0, tryptone 10.0, NaCl 10.0, and the rest water;

[0044] Solid medium was supplemented with 2% agar.

[0045] Fermentation medium (g / L): corn flour 64, soybean meal 40, amylase 2.7, Na2HPO4 4, KH2PO4 0.3, the rest is water; keep warm at 90℃ for 30min and then sterilize at 121℃ for 20min.

[0046] In the present invention, the zymogen region sequence of wild-type keratinase KER is as shown in SEQ ID Shown in NO.1: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVL GVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.

[0047] In the present invention, the zymogen region sequence of the keratinase L136A mutant is as shown in SEQ ID Shown in NO.3: MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGFKSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVAHALAQTVPYGIPLIKADKVQAQGFKGANVKVAVADTGIQASHPDLNVVGGASFVAGEAYNTDGNGHGTHVAGTVAALDNTTGVL GVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWATTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSSGNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGAAALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ.

[0048] The present invention will be further explained below through specific examples.

[0049] Example 1 Obtaining wild-type keratinase gene

[0050] 1. Use the OMEGA Bacterial DNA Kit to extract genomic DNA of Bacillus licheniformis ATCC14580. The extraction steps are as follows:

[0051] (1) Inoculate the strain onto an LB solid plate using an inoculation loop and culture at 37°C overnight.

[0052] (2) Pick a single colony from the culture plate and inoculate it into liquid test tube culture medium, and culture it at 37℃ and 220r / min with shaking overnight.

[0053] (3) Place 3 mL to 5 mL of bacterial solution in a sterilized EP tube, centrifuge at 12,000 rpm for 2 min, and discard the supernatant.

[0054] (4) Add 200 μL of sterile water to the EP tube to resuspend the bacteria, then add 50 μL of lysozyme, pipette to mix, and keep at 37°C for 20 min.

[0055] (5) Add 100 μL of BTL buffer and 20 μL of proteinase K to the EP tube, vortex and mix, and incubate at 55°C for 40 min, vortexing and mixing every 20 min.

[0056] (6) Add 5 μL of RNase, invert and mix several times, and let stand at room temperature for 10 minutes.

[0057] (7) Centrifuge at 12000 rpm for 2 min to remove the undigested portion. Transfer the supernatant to a new EP tube, add 220 μL of BDL buffer, and incubate in a 65°C water bath for 15 min.

[0058] (8) Add 220 μL of anhydrous ethanol and mix thoroughly by pipetting.

[0059] (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. Repeat twice and discard the waste liquid.

[0060] (10) Add 500 μL HBC buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate.

[0061] (11) Add 700 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 rpm for 1 min, and discard the filtrate.

[0062] (12) Add 500 μL DNA wash buffer, let stand for 1 min, centrifuge at 12000 rpm for 1 min, and discard the filtrate.

[0063] (13) Run at 12000 r / min for 2 min, discard the waste liquid tube, and place the recovery column in a new EP tube.

[0064] (14) Place in a 55°C metal bath and dry for 10 minutes.

[0065] (15) Add 50 μL of 55°C sterile water, let it stand at room temperature for 5 min, centrifuge at 12,000 rpm for 2 min, discard the recovery column, and the liquid in the EP tube is the genome.

[0066] 2. Using the extracted genome of Bacillus licheniformis 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 the present invention are as follows:

[0067] Upstream primer P1:

[0068] 5'-CGCGGATCC ATGATGAGGAAAAAAGAGTTTTTGGCT-3'

[0069] Downstream primer P2:

[0070] 5'-TCCCCCGGGTTAGTGATGATGATGATGATGTTGAGCGGCACCTTCGA-3'

[0071] P1 and P2 were used as upstream and downstream primers, and the Bacillus licheniformis genome was used as a template for amplification.

[0072] The amplification reaction system is:

[0073] Upstream primer P1 2.0 μL Downstream primer P2 2.0 μL DNA template 2.0 μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 19 μL

[0074] The amplification program was as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 57℃ annealing for 20s, 72℃ extension for 6s, 30 cycles; 72℃ extension for 10min. The PCR amplification product was electrophoresed on 0.8% agarose gel to obtain a 1140bp band ( Figure 1 ), and the PCR product was recovered using a small amount of DNA recovery kit to obtain the wild-type keratinase zymogen region gene bliker (SEQ ID NO. 2) of the present invention.

[0075] The bliker and pBSA43 plasmids were double-digested with restriction enzymes BamHI and SmaI, respectively. The bliker recovered from the gel was ligated with the vector pBSA43 to obtain the recombinant plasmid pBSA43-bliker. The enzyme digestion was verified as follows: Figure 2 As shown, it was transformed into Escherichia coli JM109 and Bacillus subtilis WB600 to obtain the recombinant Bacillus subtilis WB600 / pBSA43-bliker.

[0076] Example 2 Construction of a keratinase mutant library to screen for keratinase mutants with enhanced keratinase activity

[0077] 1. Site-directed mutagenesis was performed based on overlapping PCR technology to construct a new keratinase. The mutation primers were designed as follows:

[0078]

[0079] In the first step of overlapping PCR reaction system, P1 was used as the upstream primer, 136-R was used as the downstream primer, and plasmid pBSA43-bliker was used as the template to carry out PCR1 reaction to obtain the upstream fragment; P2 was used as the upstream primer, 136-F was used as the downstream primer, and plasmid pBSA43-bliker was used as the template to carry out PCR1 reaction to obtain the downstream fragment.

[0080] The reaction system for upstream fragment amplification is:

[0081] P1 2μL 136-R 2μL Plasmid pBSA43-bliker 2μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 19 μL

[0082] The reaction system for downstream fragment amplification is:

[0083] P2 2μL 136-F 2μL Plasmid pBSA43-bliker 2μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 19 μL

[0084] The amplification program was as follows: 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 at 72°C for 6 s; and extension at 72°C for 10 min.

[0085] 2. After gel excision and recovery of upstream and downstream fragments, PCR2 was performed. The reaction system was:

[0086] Upstream fragment 2.0 μL Downstream fragment 2.0 μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 17μL

[0087] The amplification program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 57°C for 20 s, and extension at 72°C for 6 s, for 5 cycles; and extension at 72°C for 10 min.

[0088] After PCR 2, 2 μL of primers P1 and P2 were added to the system. PCR 3 was performed using the following protocol: pre-denaturation at 98°C for 30 seconds, followed by 30 cycles of denaturation at 98°C for 10 seconds, annealing at 57°C for 20 seconds, and extension at 72°C for 6 seconds, and finally extension at 72°C for 10 minutes. The PCR product was subjected to 0.8% agarose gel electrophoresis and recovered using a mini DNA recovery kit to obtain the keratinase site-directed mutant gene, blikerL136A.

[0089] 4. The keratinase site-directed mutant gene blikerL136A was ligated to the expression vector pBSA43 and transformed into Escherichia coli JM109. The plasmid was extracted to obtain the recombinant plasmid pBSA43-blikerL136A.

[0090] The recombinant plasmid pBSA43-blikerL136A was then transformed into Bacillus subtilis WB600 to obtain the recombinant strain WB600 / pBSA43-blikerL136A. The subtilis transformants were activated onto a newly streaked Kan plate and incubated inverted at 37°C for 12 hours. The mutant strains were then screened as follows:

[0091] (1) Under sterile conditions, single mutant colonies and wild-type colonies (i.e., WB600 / pBSA43-bliker) were picked and inoculated into 5 mL of liquid LB tubes containing Kan resistance, and cultured overnight at 37°C and 220 rpm.

[0092] (2) Pipette 1 mL of bacterial solution from the test tube and add it to 50 mL of liquid LB medium containing Kan resistance. Incubate at 37°C and 220 rpm for 48 h.

[0093] (3) After the culture is completed, remove the flask and measure the bacterial concentration of the bacterial solution at OD600.

[0094] (4) Collect the bacterial solution into a 50 mL centrifuge tube and centrifuge it at 8000 rpm for 10 min. The supernatant is used as the enzyme solution for enzyme activity determination.

[0095] 5. Determination of keratinase activity by national standard method (Folin phenol method):

[0096] Keratinase hydrolyzes keratin at a certain temperature and pH (unless otherwise specified, the temperature in this invention is 60°C and pH 10), producing amino acids containing phenolic groups. These amino acids are reduced with Folin-phenol reagent to produce tungsten blue, and the absorbance of the solution is measured at 680 nm using a UV spectrophotometer. The enzyme activity is proportional to the absorbance, and the specific activity of the keratinase can be calculated. The determination method is as follows:

[0097] To the blank group, 1 mL of enzyme solution was added and kept warm at 60°C for 2 min. 2 mL of trichloroacetic acid was added and the mixture was reacted at 60°C for 10 min. 1 mL of keratin (10 g / L) solution was added and the mixture was taken out and allowed to stand for 10 min. The mixture was centrifuged at 12000 r / min for 2 min. 0.5 mL of supernatant was added and 2.5 mL of Na2CO3 was added. 0.5 mL of Folin phenol reagent was added and the mixture was color developed at 60°C for 20 min. The absorbance of the solution was measured at 680 nm using a UV spectrophotometer with a 10 mm cuvette.

[0098] Add 1 mL of enzyme solution to the sample group, keep it at 60℃ for 2 min, add 1 mL of keratin (10 g / L) solution, react at 60℃ for 10 min, add 2 mL of trichloroacetic acid, take out and let it stand for 10 min, centrifuge at 12000 r / min for 2 min, take 0.5 mL of supernatant and add 2.5 mL of Na2CO3, add 0.5 mL of folin phenol reagent, develop color at 60℃ for 20 min, and measure the absorbance of the solution at 680 nm using a UV spectrophotometer with a 10 mm cuvette.

[0099] Subtract the OD value of the blank group from the OD value of the sample group to obtain ΔOD, and then substitute ΔOD into the following formula to calculate the corresponding enzyme activity:

[0100] (N is the dilution factor of the sample)

[0101] At the same time, the mutant strain plasmid pBSA43-blikerL136A was sent to GENEWIZ for sequencing. After confirming that the mutation site was correct, the encoding gene of the keratinase mutant in which the amino acid Leu at position 136 was mutated to Ala was named blikerm1.

[0102] After enzyme activity assay, we found that the mutant L136A had higher keratinase activity than the wild type at 60°C. The enzyme activity is shown in the following table:

[0103] Keratinase Enzyme activity (U / mL) Specific activity (U / mg) WT 1404.9 780.5 L136A 2031.7 1069.4

[0104] Example 3 Expression and Preparation of Keratinase Mutants in Recombinant Bacillus amyloliquefaciens Strains

[0105] The wild-type KER encoding gene bliker and the mutant L136A encoding gene blikerm1 were ligated with the Bacillus amyloliquefaciens expression plasmid pBSA43 to obtain new recombinant plasmids pBSA43-bliker and pBSA43-blikerm1, respectively.

[0106] The recombinant plasmids pBSA43-bliker and pBSA43-blikerm1 were respectively transformed into Bacillus amyloliquefaciens CGMCC No.11218. After kanamycin (Kan) resistance screening and enzyme digestion verification, the wild-type recombinant strain CGMCC No.11218 / pBSA43-bliker and the mutant recombinant strain CGMCC No.11218 / pBSA43-blikerm1 were obtained.

[0107] The recombinant strain CGMCC No.11218 / pBSA43-blikerm1 of Bacillus amyloliquefaciens and the wild-type recombinant strain CGMCC No.11218 / pBSA43-bliker were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 μg / mL), cultured at 37°C, 220 r / min overnight, transferred to 50 mL of fresh fermentation medium (containing kanamycin, 50 μg / mL) according to a 2% inoculum amount, and continued to be cultured at 37°C, 220 r / min for 48 h.

[0108] Fermentation medium (g / L): corn flour 64, soybean meal 40, amylase 2.7, Na2HPO4 4, KH2PO4 0.3, the rest is water; keep warm at 90℃ for 30min and then sterilize at 121℃ for 20min.

[0109] The fermentation broth was centrifuged and the supernatant was measured for enzyme activity. The keratinase activity obtained from the fermentation of Bacillus amyloliquefaciens was measured using the national standard method in Example 2. The keratinase activity of wild-type KER in Bacillus amyloliquefaciens was 3512.3 U / mL, and the activity of mutant L136A was 5025.9 U / mL.

[0110] Preparation of pure protease powder: The supernatant obtained by centrifugation of the fermentation broth prepared above is first salted out with ammonium sulfate at 25% saturation to remove impurities, then the saturation is increased to 65% to precipitate the target protein. After dissolution, the supernatant is dialyzed for desalination, and the active component obtained after dialysis and desalination is dissolved in 0.02 mol / L Tris-HCl (pH 7.0) buffer. The sample is applied to a cellulose ion exchange chromatography column and the unadsorbed protein is first eluted with the same buffer. Then, a gradient elution is performed using 0.02 mol / L Tris-HCl (pH 7.0) buffer containing different concentrations of NaCl (0-1 mol / L) to collect the target protein. The active component obtained by ion exchange is first equilibrated with 0.02 mol / L Tris-HCl (pH 7.0) buffer containing 0.15 mol / L NaCl, applied to a Sephadex G25 gel chromatography column and eluted with the same buffer at a rate of 0.5 mL / min to obtain a purified enzyme solution. After freeze-drying, the pure keratinase powder is obtained.

[0111] The prepared keratinase mutant enzyme powder can be applied to the fields of leather making, food, feed, etc.

[0112] Example 4 Application of Keratinase for Wool Hydrolysis

[0113] Wool, a natural protein fiber, is widely used in the textile industry for its excellent properties, including warmth, softness, and easy dyeing. However, due to the presence of scales in the wool fiber structure, the fabric often shrinks and deforms after washing, causing a tingling sensation during wear. To overcome this shortcoming, keratinase is used during processing to remove the scales and improve the performance of wool fabrics.

[0114] Wool slivers were washed and dried at 60°C. Equal masses of wool (1 g) were weighed and placed in 50 mL of Gly-NaOH buffer. 0.1 g of wild-type keratinase and mutant L136A were added, respectively. Both were reacted with untreated wool slivers in a shaking waterbath at 40°C for 6 hours. The enzymes were then inactivated by incubation at 85°C for 10 minutes, washed, and dried at 60°C. The UV absorption spectrum of the hydrolysis residue after enzyme treatment was measured using a UV-visible spectrophotometer, and the effect on wool fibers was analyzed based on the absorbance at 280 nm.

[0115] Keratinase successfully breaks the disulfide bonds in the wool fiber scale layer and further hydrolyzes the cleaved protein peptides into amino acids that dissolve in the solution, resulting in an increase in absorbance. The results showed that the absorbance of the hydrolyzate of untreated wool at 280nm was 0.14, the absorbance of the hydrolyzate of wool treated with wild-type keratinase was 0.33, and the absorbance of the hydrolyzate of wool treated with mutant L136A was 0.58. Since the absorbance of the solution after treating wool tops with mutant L136A was significantly higher than that after treating with wild-type keratinase, that is, the amino acid content in the solution increased, indicating that mutant L136A more effectively degrades the wool scale layer, improves the efficiency of wool scale hydrolysis, and improves the performance of wool products.

[0116] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details 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 keratinase mutant is obtained by causing L136A mutation on the basis of the wild-type keratinase shown in SEQ ID NO.

1. The amino acid sequence of the keratinase mutant is shown in SEQ ID NO.

3.

2. The gene encoding the keratinase mutant according to claim 1.

3. A recombinant plasmid or recombinant strain comprising the mutant encoding gene according to claim 2.

4. Use of the recombinant plasmid or recombinant strain according to claim 3 in producing the keratinase mutant according to claim 1.

5. The use of the keratinase mutant according to claim 1, characterized in that: It is used in hydrolyzed keratin.

6. The use according to claim 5, characterized in that It is used in feed processing industry, leather industry or cosmetics industry.

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