Keratinase mutant and its application

Abstract

The invention belongs to the field of bioengineering technology, and is specifically related to keratinase mutants and encoding genes and applications. The keratinase mutant is obtained by subjecting the wild-type keratinase derived from Bacillus subtilis SJ-215 to L103I, L129S, V177A, S231A, and V311A mutations, and has the amino acid sequence shown in sequence table SEQ ID NO.2. The keratinase after mutation has good alkali resistance and heat resistance, and the optimum reaction pH is 8.5. After being treated for 20h under the conditions of pH 6.5-12.5, the relative enzyme activity still remains at more than 80%, and the optimum reaction temperature is 55 DEG C., and is kept warm for 20h under the conditions of 40-75 DEG C, and more than 90% of the enzyme activity can still be retained, and the wool fabric felting rate after treatment is 6%, which is particularly suitable for the anti-felting treatment of wool fabric.

Description

Technical field:

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a keratinase mutant and a coding gene and application thereof. Background technology:

[0002] Keratinase is a specific hydrolase produced by microorganisms that can specifically degrade keratin substrates, such as wool, feathers, and cattle and sheep horns. Most keratinases are serine proteases, and a small number are metalloproteases and aspartic proteases. Keratinase-producing microorganisms mainly come from bacteria, fungi, actinomycetes, etc., and most of them are screened from feather piles or hair piles. Among bacteria, the main strains that produce keratinase are Bacillus, such as the first reported Bacillus licheniformis PWD-1 that can secrete keratinase and degrade feathers. In addition, there are Bacillus subtilis, Bacillus brevis, Bacillus cereus, etc.

[0003] In the textile industry, wool fabric dyeing and finishing process, due to the shrinkage of wool, wool fabric will feel felt when subjected to external force in a hot and humid state, which will cause the fabric area to shrink, shape to change, fluff to protrude, elasticity to decrease, and feel rough, seriously affecting the appearance and wearing performance of the fabric. Therefore, it is necessary to perform anti-felting finishing on wool fabric.

[0004] There are two main methods for anti-felting of wool fabrics: reduction anti-felting with the main purpose of removing scales (the main component is keratin) and addition anti-felting by coating the wool fiber surface with polyurethane or silicone polymer resin. At present, the most widely used method for anti-felting of wool is the chlorination method in reduction anti-felting, such as treatment with hypochlorite or dichloroisocyanurate (DCCA). However, wool fibers are prone to yellowing due to chlorination treatment, the fabric feels rough, and it is easy to be uneven when dyed. In addition, the content of organic chloride (AOX) in the wastewater after treatment is quite high, which has an adverse effect on the environment and has become less and less popular in the industry. A large number of studies have also been conducted on biological anti-felting of wool fabrics using proteases. When using proteases to anti-felting wool, it is generally necessary to chemically pretreat the wool surface, such as oxidation pretreatment with hydrogen peroxide, potassium permanganate, etc., to increase the hydrophilicity of the fiber surface, and then treat it with proteases to remove the scale layer on the wool surface. Although conventional chemical oxidation pretreatment can increase the weight-reducing effect of protease, it is difficult to control the uniformity of fiber scale layer treatment, which can easily cause internal damage to the wool fibers, resulting in increased weight loss, decreased strength, and little improvement in anti-felting effect. Therefore, the wool protease weight-reduction and anti-felting finishing process is difficult to apply in practice.

[0005] The use of keratinase for biological anti-felting of wool fabrics does not require chemical pretreatment of wool fabrics. The process of keratinase removing wool scales is as follows: first, keratinase destroys the disulfide bonds in the keratin molecules in the scales to form denatured keratin, which makes the keratin lose its ability to be insoluble in water and resist the action of proteases; then it further hydrolyzes the denatured protein to generate polypeptides, oligopeptides and free amino acids; finally, ammonia and sulfide are produced through transamination to completely hydrolyze the keratin. Cai Shaobo et al. isolated a bacterium Pseudomonas with the ability to degrade protein from rotten wool fabrics. The keratinase secreted by the bacterium can improve the anti-felting performance and tensile strength of wool and polyester blended fabrics. Keratinase from Bacillus thuringiensis L11 and Bacillus licheniformis can improve the dyeing performance and felting resistance of wool without losing fibers, thereby improving the quality of wool.

[0006] Since the alkaline wool washing method is the most widely used in the wool washing stage before the wool anti-felting treatment, the temperature of the wool washing tank in this method is 50-60℃, not exceeding 75℃, and the pH of the washing liquid is controlled at 8-11. The subsequent anti-felting treatment requires a temperature of 50-60℃ and a pH of 8-11 to minimize energy consumption and save costs. However, the optimal reaction pH of the main keratinase is currently 7-7.5, and the optimal reaction temperature is 45℃. The enzyme has poor stability when the pH is higher than 8.0 or the temperature is higher than 55℃. In addition, the fermentation enzyme activity level of keratinase is low, resulting in high production costs, which also limits its large-scale application.

[0007] With the promotion of green industry, the industrialization of keratinase is urgently needed, but the degradation efficiency of keratin in fabrics and the acid, alkali and heat resistance of keratinase still need to be improved. With the continuous advancement of science and technology and the requirements of the downstream textile industry for the low-cost application of keratinase, it is necessary to further upgrade the enzymatic properties and fermentation enzyme activity of keratinase to adapt to the changing market demand. Summary of the invention:

[0008] The main purpose of the present invention is to provide a keratinase mutant and its gene, a recombinant expression vector and a recombinant strain containing the gene, and a method for preparing keratinase by the recombinant bacteria, aiming to solve the problems in the prior art that keratinase has poor alkali resistance and heat resistance, low fermentation enzyme activity level, low degradation efficiency of keratin in wool fabrics, and poor resistance to wool fabric felting shrinkage.

[0009] To achieve the above object, the present invention obtains a keratinase mutant by mutating leucine, leucine, valine, serine and valine at positions 103, 129, 177, 231 and 311 in the amino acid sequence of a wild-type keratinase derived from Bacillus subtilis SJ-215 (deposit number CGMCC NO.18645) to isoleucine, serine, alanine, alanine and alanine, respectively. The amino acid sequence of the mutant is shown in the sequence table SEQ ID NO.2.

[0010] The present invention also provides a gene encoding the mutant;

[0011] Furthermore, the nucleotide sequence of the coding gene is shown in the sequence listing SEQ ID NO.4.

[0012] The present invention also provides the application of the protease mutant, especially the application in degrading keratin substrates, more especially the application in anti-felting finishing of wool fabrics in the textile industry.

[0013] The present invention also provides an integration expression vector, comprising the above-mentioned keratinase mutation encoding gene;

[0014] Preferably, the expression plasmid used in the integration expression vector is pKS1;

[0015] The present invention also provides a recombinant strain, comprising the above-mentioned keratinase mutation encoding gene or recombinant expression vector;

[0016] Preferably, the host cell of the strain is Bacillus amyloliquefaciens ZD-708 (ΔGAΔEGL), which has been disclosed in Chinese invention patent CN 201911119213.5.

[0017] The present application also provides the application of the above-mentioned recombinant expression vector or recombinant strain, especially the application in preparing the keratinase shown in SEQ ID NO.2;

[0018] Furthermore, the method for preparing keratinase using the above recombinant strain is as follows:

[0019] At 60m 3 Culture in fermenter: Inoculate 2-4% of the volume of the fermenter medium, culture temperature 33-36°C, tank pressure 0.04-0.08MPa, initial speed 100rpm, initial air volume 200m 3 / h, the dissolved oxygen is controlled to be greater than 30% by increasing the speed and ventilation volume, the maximum speed is 600rpm, and the maximum ventilation volume is 4000m 3 / h; when the fermentation reaches 10h-15h and the rotation speed and air volume of the fermentation tank reach the maximum, 30% glucose is added to control the dissolved oxygen at 40%-50%, and ammonia is added to control the pH at 6.8-7.0. The fermentation is terminated when the bacteria autolysis is serious. The culture period is 90-100h.

[0020] Furthermore, in 3m 3 The culture conditions in the seed tank are: 3-10% inoculation amount, temperature 33-36°C, tank pressure 0.04-0.08Mpa, initial speed 100rpm, initial air volume 20m 3 / h, the dissolved oxygen is controlled to be greater than 30% by increasing the speed and air volume, the maximum speed is 600rpm, and the maximum ventilation volume is 200m 3 / h, add ammonia to control pH at 6.8-7.0, and culture for 48h;

[0021] Further, the seed culture medium is composed of: tryptone 12-18 g / L, yeast extract 1-3 g / L, potassium chloride 0.20-0.42 g / L, calcium chloride 0.18-0.35 g / L, magnesium sulfate 2.5-3.2 g / L, maltose 2.1-3.2 g / L, soy peptone 4.5-8.0 g / L, potassium dihydrogen phosphate 1.4-1.6 g / L, disodium hydrogen phosphate 0.7-1.26 g / L, pH 6.8-7.0;

[0022] Furthermore, the fermentation medium is composed of: 3-6 g / L corn flour, 2-4 g / L soy peptone, 3-5 g / L soybean cake powder, 1.2-1.8 g / L yeast powder, 1.3-2.5 g / L corn steep liquor, 2.5-3.0 g / L disodium hydrogen phosphate, 1.7-2.4 g / L potassium dihydrogen phosphate, and pH is 6.8-7.0.

[0023] Beneficial effects:

[0024] The keratinase mutant provided by the present invention has an optimum reaction pH of 8.5. After being treated at pH 6.5-12.5 for 20 hours, the relative enzyme activity is still maintained at more than 80%. The optimum reaction temperature is 55°C. After being kept at 40-75°C for 20 hours, the enzyme activity can still be retained at more than 90%. The felting rate of wool fabric treated by the mutant is 6%. The enzymatic characteristics are particularly suitable for anti-felting treatment of wool fabric.

[0025] The keratinase mutant provided by the present invention is integrated into the genome of the host bacteria for expression, thereby preventing the mutant gene from being lost in the host bacteria and enabling the mutant gene to be stably expressed in the host bacteria at 60 m 3The average fermentation enzyme activity in the fermentation tank is 30186U / mL, which is 1.6 times higher than that of the wild-type enzyme. It can further reduce the production cost of keratinase, promote the widespread application of keratinase in the textile industry, and actively respond to the dilemma of rising raw material prices.

[0026] In addition, the present invention confirms 5 amino acid sites in keratinase that are related to the heat resistance and high efficiency degradation of keratin in wool, and proves the importance of these sites to the enzyme's heat resistance and high efficiency degradation of keratin in wool fabric to reduce the felt shrinkage of wool fabric. This provides important clues for studying the heat resistance of keratinase and the high efficiency degradation of keratin in wool fabric to reduce the felt shrinkage of wool fabric, and also provides a reliable reference for the research of other keratinases. Description of the drawings:

[0027] Figure 1 PCR verification of the recombinant strain ZD-708(△GAΔEGL)-K12;

[0028] Figure 2 The optimal reaction pH curve of keratinase before and after mutation;

[0029] Figure 3 pH stability range curve of keratinase before and after mutation;

[0030] Figure 4 The optimal reaction temperature curve of keratinase before and after mutation;

[0031] Figure 5 Temperature stability range curve of keratinase before and after mutation. Specific implementation method:

[0032] The present invention is described in detail below through specific implementation cases. The specific implementation cases are only for illustration and are not intended to limit the scope of implementation of the present invention.

[0033] According to the technical solution of the present invention, the wild-type keratinase is derived from Bacillus subtilis SJ-215, and its deposit number is CGMCC NO.18645.

[0034] The keratinase mutant of the present invention is obtained by subjecting the amino acid sequence of the wild-type keratinase to point mutations L103I, L129S, V177A, S231A and V311A. The amino acid sequence of the keratinase mutant L103I / L129S / V177A / S231A / V311A is shown in SEQ ID NO.2.

[0035] The identification of the keratinase mutant used in the present invention:

[0036] The mutated amino acid in the mutant is represented by "amino acid replaced by the original amino acid position". For example, L129S means that the amino acid at position 129 is replaced by Ser from the wild-type Leu, and the position number corresponds to the amino acid sequence number of the wild-type keratinase in SEQ ID NO.1, as shown in the following table.

[0037]

[0038] The specific sequence information is as follows:

[0039] SEQ ID NO.1

[0040] MMRKKSFWLGMLTALMLVFTIVGFKSGVKTASVKKDIIKEKEARVAPIKEVKNDPDVAYVE

[0041] EDHMAFSDSASAAQPAKNVEKDYSGGKVDKQFRIINAAKAKLDQAVDNAYARGVVVVAA

[0042] AGNSVAHALAQTVPYGIPLIKADKYNTDGNGHGTHVAGTVAALDVQAQGFKGANVKVAV

[0043] LDTGILNGTSMASPHVAGAAALILSNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWA

[0044] TTQASHPDLNVVGGASFVAGEANGMDVINMSLGGASGSTAMKGSSGNTMAFNTIGYPAK

[0045] YDSVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATKHPNLSASQVRNRLS

[0046] STATYLGSSFYYGKGLINVEAAAQ

[0047] SEQ ID NO.2

[0048] MMRKKSFWLGMLTALMLVFTIVGFKSGVKTASVKKDIIKEKEARVAPIKEVKNDPDVAYVE

[0049] EDHMAFSDSASAAQPAKNVEKDYSGGKVDKQFRIINAAKAKIDQAVDNAYARGVVVVAA

[0050] AGNSVAHASAQTVPYGIPLIKADKYNTDGNGHGTHVAGTVAALDVQAQGFKGANVKAAV

[0051] LDTGILNGTSMASPHVAGAAALILSNTTGVLGVAPSVSLYAVKVLNSSGSGAYSGIVSGIEWA

[0052] TTQASHPDLNVVGGASFVAGEANGMDVINMSLGGASGSTAMKGSSGNTMAFNTIGYPAK

[0053] YDSVIAVGAADSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATKHPNLSASQVRNRLS

[0054] STATYLGSSFYYGKGLINVEAAAQ

[0055] SEQ ID NO.3

[0056] ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTAATGCTCGTGTTCAC

[0057] GATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCCGTCAAAAAGGACATCATCAAAG

[0058] AGAAAGAAGCGCGCGTTGCGCCAATTAAGGAAGTCAAAAATGATCCGGATGTCGCTTAT

[0059] GTGGAAGAGGATCATATGGCCTTCAGCGATTCCGCGTCTGCTGCTCAGCCGGCGAAAAA

[0060] TGTTGAAAAGGATTATAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGG

[0061] CAAAAGCGAAGCTAGACCAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGT

[0062] AGCTGCAGCAGGGAACAGCGTGGCCCATGCCTTGGCGCAAACCGTTCCTTACGGCATTC

[0063] CTCTCATTAAAGCGGACAAATATAACACCGACGGCAACGGACACGGCACACATGTTGCC

[0064] GGTACAGTAGCTGCGCTTGACGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGT

[0065] AGCCGTCCTGGATACAGGAATCTTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGG

[0066] GAGCAGCAGCTTTGATCTTGTCAAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTAT

[0067] CCTTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGATCATACAGCGGCATTGTA

[0068] AGCGGAATCGAGTGGGCGACAACACAAGCTTCTCATCCGGACTTGAACGTAGTCGGCG

[0069] GAGCAAGCTTTGTGGCTGGCGAAGCTAACGGCATGGATGTTATCAATATGAGCCTTGGG

[0070] GGAGCATCAGGCTCGACAGCGATGAAAGGATCTTCAGGAAACACGATGGCCTTCAATAC

[0071] AATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGTAGACTCTAACAG

[0072] CAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAG

[0073] GCGTATACAGCACTTACCCAACGAACACTTATGCAACAAAACATCCGAACCTTTCAGCT

[0074] TCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTAT

[0075] GGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA

[0076] SEQ ID NO.4

[0077] ATGATGAGGAAAAAGAGTTTTTGGCTTGGGATGCTGACGGCCTTAATGCTCGTGTTCAC

[0078] GATTGTCGGATTTAAGTCAGGAGTGAAAACCGCATCCGTCAAAAAGGACATCATCAAAG

[0079] AGAAAGAAGCGCGCGTTGCGCCAATTAAGGAAGTCAAAAATGATCCGGATGTCGCTTAT

[0080] GTGGAAGAGGATCATATGGCCTTCAGCGATTCCGCGTCTGCTGCTCAGCCGGCGAAAAA

[0081] TGTTGAAAAGGATTATAGCGGCGGAAAAGTGGACAAGCAGTTTAGAATCATCAACGCGG

[0082] CAAAAGCGAAGATAGACCAGGCAGTCGACAATGCATATGCAAGAGGGGTTGTCGTTGTA

[0083] GCTGCAGCAGGGAACAGCGTGGCCCATGCCTCGGCGCAAACCGTTCCTTACGGCATTCC

[0084] TCTCATTAAAGCGGACAAATATAACACCGACGGCAACGGACACGGCACACATGTTGCCG

[0085] GTACAGTAGCTGCGCTTGACGTGCAGGCTCAAGGCTTTAAGGGAGCGAATGTAAAAGC

[0086] AGCCGTCCTGGATACAGGAATCTTGAACGGAACGTCAATGGCTTCTCCTCATGTAGCGG

[0087] GAGCAGCAGCTTTGATCTTGTCAAATACAACGGGTGTATTAGGCGTTGCGCCAAGCGTAT

[0088] CCTTGTACGCGGTTAAAGTACTGAATTCAAGCGGAAGCGGAGCATACAGCGGCATTGTA

[0089] AGCGGAATCGAGTGGGCGACAACACAAGCTTCTCATCCGGACTTGAACGTAGTCGGCG

[0090] GAGCAAGCTTTGTGGCTGGCGAAGCTAACGGCATGGATGTTATCAATATGAGCCTTGGG

[0091] GGAGCATCAGGCTCGACAGCGATGAAAGGATTCTTCAGGAAACACGATGGCCTTCAATAC

[0092] AATTGGCTATCCTGCGAAATACGATTCTGTCATCGCTGTTGGTGCGGCAGACTCTAACAG

[0093] CAACAGAGCTTCATTTTCCAGTGTGGGAGCAGAGCTTGAAGTCATGGCTCCTGGCGCAG

[0094] GCGTATACAGCACTTACCCAACGAACACTTATGCAACAAAACATCCGAACCTTTCAGCT

[0095] TCACAAGTCCGCAACCGTCTCTCCAGCACGGCGACTTATTTGGGAAGCTCCTTCTACTAT

[0096] GGGAAAGGTCTGATCAATGTCGAAGCTGCCGCTCAATAA

[0097] The present invention will be further explained below in conjunction with specific implementation modes.

[0098] Example 1 Obtaining keratinase mutant gene, expression vector and recombinant strain

[0099] 1.1 Acquisition of keratinase mutant gene

[0100] The invention obtains the keratinase mutant of the invention by mutating leucine, leucine, valine, serine and valine at positions 103, 129, 177, 231 and 311 in the amino acid sequence of a keratinase derived from Bacillus subtilis SJ-215 and with a preservation number of CGMCCNO.18645 to isoleucine, serine, alanine, alanine and alanine, respectively.

[0101] 300 mL of Bacillus subtilis SJ-215 logarithmic seed solution was placed in 6 50 mL centrifuge tubes, centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the precipitated bacteria were washed twice with 600 mL of physiological saline, and then sent to Shanghai Biotech for genome sequencing to obtain gene sequence and upstream and downstream sequence information. According to the obtained genome, primers Y1 (containing HindⅢ restriction site) and Y2 (containing PstⅠ restriction site) were used to amplify the keratinase gene K11, and connected to the T vector for storage, and this recombinant vector was named T-0. The site-directed mutagenesis kit used the kit of Shanghai Biotech, model B639281, and the specific operation steps are as follows:

[0102] (1) Primers for amplifying the original keratinase gene and various point mutations.

[0103] Table 1 The primers used for the above point mutation are as follows

[0104]

[0105]

[0106] (2) Point mutation: Taking the point mutation of L103I as an example, use the two primers L103I-1 and L103I-2, and the recombinant T vector T-0 containing the original keratinase gene K11 as a template. According to the system in the pilot mutation kit, add other reagents and perform PCR amplification. After the amplification, take 10 μL of the above mutation PCR amplification product and perform 0.8% agarose gel electrophoresis detection. After the correct band is correct, add 1 μL of digestion enzyme to the mutation product, mix well, and incubate at 37°C for 60 minutes.

[0107] (3) Transformation: Add 5 μL of the mutated product to 50 μL of DH5α competent cells, mix well, and place on ice for 30 min; heat shock at 42°C for 45 s and immediately cool on ice for 10 min; add 500 μL of LB medium, culture at 180 r / min, and 37°C for 1 h; then centrifuge the culture at 10,000 r / min for 5 min, discard part of the supernatant, retain 100-150 μL of the supernatant, flick to suspend the bacteria, take all the bacterial solution to plate, and culture at 37°C overnight.

[0108] (4) Verification of positive clones: Pick a single colony and place it in 500 μL LB medium containing ampicillin resistance. After culturing at 200 r / min for 2-3 h, perform PCR identification. Send the screened positive clones for sequencing. Compare the sequencing results and select the positive clones that match the nucleotide sequence. The positive clone is the mutant containing the recombinant plasmid with the correct mutation (L103I).

[0109] (5) Mutation of other sites: Using the correctly screened mutant as a template, the primer pairs corresponding to the mutation sites in Table 1 were used one by one, and the same method as above was used to mutate each site, and finally the keratinase mutant gene shown in SEQ ID NO.4 was screened out.

[0110] The keratinase gene K12 (SEQ ID NO. 4) obtained after the above point mutations was connected to a T vector for storage and named as T-K12 vector.

[0111] The present invention achieves expression of the keratinase mutant by replacing the neutral protease gene APR gene on the genome of the host bacteria ZD-708 (ΔGAΔEGL) with the keratinase mutant encoding gene K12 of the present invention, as follows:

[0112] 1.2 Obtaining the recombinant expression vector

[0113] 1.2.1 Design of homology arm primers

[0114] According to the 700bp-100bp range of the upstream and downstream sequences of the neutral protease gene APR sequence in the genome sequence of strain ZD-708 (ΔGAΔEGL), primers for amplifying the upstream and downstream homology arms were designed. At the same time, KpnⅠ and HindⅢ restriction sites and protective bases were added to the upstream primer, and PstⅠ and NotⅠ restriction sites and protective bases were added to the downstream primer. The designed primers are named as follows:

[0115] Two pairs of primers APR-S1 / APR-X1 and APR-S2 / APR-X2 were used to amplify the upstream and downstream homology arm fragments of the neutral protease gene APR. The names of the amplified upstream and downstream homology arm fragments were APR-S (upstream homology arm) and APR-X (downstream homology arm), respectively. Primers YZ-5 and YZ-6 were used to verify whether the APR gene was replaced by the keratinase mutant gene K12. If the APR gene fragment could be amplified, the gene was not replaced. If the K12 gene fragment could be amplified, the gene was replaced successfully.

[0116] The specific primer sequences are shown in the following table:

[0117]

[0118] 1.2.2 Amplification of homology arms and construction of recombinant plasmid pKS1-APR-SX carrying upstream and downstream homology arms of APR gene

[0119] The above two pairs of primers were used to amplify different homology arms using the genome of strain ZD-708 (ΔGAΔEGL) extracted by the kit as a template. After the amplified homology arm fragments were verified by nucleic acid electrophoresis, the two homology arm fragments APR-S and APR-X were recovered using a gel excision recovery kit. The homology arm APR-S and plasmid pKS1 were double-digested with KpnⅠ and HindⅢ, and then connected with DNA ligase to obtain the recombinant plasmid pKS1-APR-S. The homology arm APR-X and plasmid pKS1-APR-S were double-digested with PstⅠ and NotⅠ, and then connected with DNA ligase to obtain the recombinant plasmid pKS1-APR-SX.

[0120] 1.2.3 Construction of recombinant expression plasmid pKS1-APR-S-K12-X

[0121] The T-K12 vector with the keratinase mutant gene sequence and the recombinant plasmid pKS1-APR-SX were double-digested with HindⅢ and PstⅠ, and after the nucleic acid was recovered by electrophoresis gel, they were connected together with DNA ligase to form the recombinant expression plasmid pKS1-APR-S-K12-X.

[0122] 1.3 Obtaining recombinant strains

[0123] After preparing strain ZD-708 (ΔGAΔEGL) into electrocompetent cells, the recombinant expression plasmid pKS1-APR-S-K12-X was electrotransformed into strain ZD-708 (ΔGAΔEGL) by electrotransformation, and then 100 μL was spread on an erythromycin (100 μg / mL) resistant LB plate, placed in a 30°C incubator, and cultured for 15 hours. Pick a single colony grown on the resistant plate, inoculate it into 50 mL of LB liquid culture medium without resistance, place it in a shaker at 37°C, 200 rpm and culture it for 15 hours, take 100 μL and spread it on an erythromycin (100 μg / mL) resistant LB plate, place it in a 37°C incubator, and culture it for 15 hours. The single colony that grew is the transformant that has undergone homologous recombination single exchange. Then transfer the single exchange transformant to LB medium without resistance, place it in a shaker at 30°C and 200rpm for 38h, take 100μL and spread it on a LB plate without resistance, place it in a 37°C incubator and culture it for 15h, randomly select about 100 single colonies and transfer them to LB plates with erythromycin (100μg / mL) resistance, select single colonies that do not grow on the resistance plate for colony PCR identification, and use primers YZ-5 and YZ-6 for PCR amplification. If the APR gene is successfully replaced by the K12 gene, the length of the PCR product is 2580bp, and if the APR gene is not successfully replaced, the length of the PCR product is 3530bp. The strain PCR verification electrophoresis after successful replacement is as follows Figure 1 As shown, the strain in which the K12 gene was successfully integrated into the genome was named ZD-708 (ΔGAΔEGL)-K12.

[0124] Example 2 Preparation of keratinase

[0125] 2.1 Preparation of keratinase mutants by fermentation of recombinant strains

[0126] The recombinant strain ZD-708 (ΔGAΔEGL)-K12 constructed in Example 1 was used for fermentation preparation, and the method was as follows:

[0127] 3m 3 Seed culture in seed tank: inoculation amount 3%, temperature 35℃, tank pressure 0.05Mpa, initial speed 100rpm, initial air volume 20m 3 / h, the dissolved oxygen is controlled to be greater than 30% by increasing the speed and air volume, the maximum speed is 600rpm, and the maximum ventilation volume is 200m 3 / h, ammonia was added to control the pH at 6.9, and the culture was carried out for 48h;

[0128] Seed culture medium: tryptone 16g / L, yeast extract 2g / L, potassium chloride 0.30g / L, calcium chloride 0.22g / L, magnesium sulfate 2.8g / L, maltose 2.9g / L, soy peptone 7.0g / L, potassium dihydrogen phosphate 1.5g / L, disodium hydrogen phosphate 1.1g / L, the rest is water, pH 6.9;

[0129] 60m 3 Fermentation in the fermenter: Inoculate 3% of the volume of the fermenter medium, culture temperature 35°C, tank pressure 0.05MPa, initial speed 100rpm, initial air volume 200m 3 / h, the dissolved oxygen is controlled to be greater than 30% by increasing the speed and ventilation volume, the maximum speed is 600rpm, and the maximum ventilation volume is 4000m 3 / h; when the fermentation reaches 11h and the rotation speed and air volume of the fermentation tank reach the maximum, 30% glucose is added to control the dissolved oxygen at 40%-50%, and ammonia is added to control the pH at 6.9. The fermentation is terminated when the bacteria autolysis is serious. The culture period is 90-100h.

[0130] The fermentation tank medium composition is: corn flour 5g / L, soy peptone 3g / L, soybean cake powder 4g / L, yeast powder 1.6g / L, corn steep liquor 1.9g / L, disodium hydrogen phosphate 2.9g / L, potassium dihydrogen phosphate 1.8g / L, the rest is water, pH is 6.9;

[0131] The results of three batches of fermentation are as follows, with an average fermentation enzyme activity of 30186 U / mL.

[0132] batch Fermentation cycle (h) Fermentation enzyme activity (U / mL) 1 92 30160 2 90 30210 3 100 30190

[0133] 2.2 Preparation of keratinase solution produced by wild-type strain

[0134] The wild-type K11 gene (shown in SEQ ID NO.3) was constructed into the strain ZD-708 (ΔGAΔEGL) according to the method of Example 1, and ZD-708 (ΔGAΔEGL)-K11 was obtained after screening. Three batches of fermentation were carried out according to the same fermentation method as in Example 2.1. The results were as follows, and the average fermentation enzyme activity was: 11534 U / mL.

[0135] batch Fermentation cycle (h) Fermentation enzyme activity (U / mL) 1 88 11260 2 95 13112 3 98 10230

[0136] Example 3 Method for determining keratinase activity

[0137] 3.1 Definition of keratinase activity involved in the present invention

[0138] In the determination system of 3.2, at 40°C and pH 8.0, 1 μg of tyrosine is degraded and released from a keratin solution with a concentration of 10.0 g / L per minute, which is 1 enzyme activity unit, expressed as U / mL.

[0139] 3.2 Determination of keratinase activity

[0140] 3.2.1 Dilution of samples

[0141] Take an appropriate amount of supernatant (enzyme activity assay sample) and dilute it with boric acid buffer (pH 8.0, take 50 mL of 0.0038 g / mL sodium borate solution and adjust the pH to 8.0 with 0.01237 g / mL boric acid solution) to control the keratinase activity in the diluted enzyme solution to be tested between 10 U / mL and 15 U / mL.

[0142] 3.2.2 Drawing of L-tyrosine standard curve

[0143] Color reaction: Accurately pipette 1.0 mL of each L-tyrosine standard series solution in the table below, put it into a stoppered test tube (do 2 parallels for each concentration), add 5.0 mL of 0.4 mol / L sodium carbonate solution and 1.0 mL of Folin phenol reagent, shake well. Place it in a 40℃ water bath to develop color for 20 minutes, take it out, place it in cold water, quickly cool it to room temperature, use a 10mm colorimetric dish, use tube 0 without tyrosine as blank, and measure its absorbance at a wavelength of 680nm on a spectrophotometer.

[0144] L-Tyrosine standard stock solution (1 mg / mL): Accurately weigh 0.1 g of L-tyrosine standard substance (purity ≥ 95%) that has been dried at 105°C for 4 h to constant weight, accurate to 0.000 1 g, dissolve it in 20 mL of 1 mol / L hydrochloric acid solution, then dilute to 100 mL with water and mix well.

[0145] L-Tyrosine standard working solution (100 μg / mL): Before use, accurately pipette 10 mL of 1 mg / mL L-Tyrosine standard stock solution, dilute to 100 mL with 0.1 mol / L hydrochloric acid solution, and mix well.

[0146] L-Tyrosine Standard Series Solution

[0147]

[0148] Standard curve drawing: Draw the standard curve with absorbance A as the horizontal axis and L-tyrosine concentration c as the vertical axis. The standard curve calculation formula is c = 100 × A. Its K value should be in the range of 95 to 105, and the regression coefficient R 2 It can be used only when the value is above 0.998. If it does not meet the requirements, the reagent needs to be re-prepared and tested.

[0149] Note: The L-tyrosine standard working solution should be assayed immediately after preparation.

[0150] 3.2.3 Determination steps and result calculation

[0151] 3.2.3.1 Preparation of 1% water-soluble keratin solution

[0152] Use Gly / NaOH solution with pH 8.0 as buffer to prepare water-soluble keratin with a concentration of 1% (w / v) (purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd., product code: K0043). This solution is stored in a refrigerator at 4°C and has a shelf life of 3 days.

[0153] 3.2.3.2 Determination of blank control

[0154] Place 1% water-soluble keratin solution in a 40℃ constant temperature water bath and preheat for 5 minutes. Pipette 1.00mL of diluted enzyme solution into a 10mL test tube and place in a 40℃ water bath for 2 minutes. Add 2.00mL of 0.4mol / L trichloroacetic acid solution, shake for 3s, and keep warm at 40℃ for 10 minutes. Add 1.00mL of preheated water-soluble keratin solution and mix well. Let stand at room temperature for 10 minutes and filter with filter paper. Take 1.00mL of filtrate, add 5.00mL of 0.4mol / L sodium carbonate solution and 1.00mL of Folin phenol reagent, and keep warm at 40℃ for 20 minutes to develop color. At a wavelength of 680nm, use a 10mm cuvette to measure the absorbance (A0), and calculate the L-tyrosine concentration c0 using the standard curve formula.

[0155] 3.2.3.3 Sample determination

[0156] Pipette 1.00mL of the diluted enzyme solution into a 10mL test tube and place in a 40℃ water bath for 2min. Add 1.00mL of preheated (40℃) 1% water-soluble keratin solution, shake for 3s, and keep warm at 40℃ for 10min. Add 2.00mL of 0.4mol / L trichloroacetic acid solution and mix well. Let stand at room temperature for 10min and filter with filter paper. Take 1.00mL of the filtrate, add 5.00mL of 0.4mol / L sodium carbonate solution and 1.00mL of Folin phenol reagent, and keep warm at 40℃ for 20min to develop color. Measure the absorbance (A) at a wavelength of 680nm using a 10mm cuvette, and calculate the L-tyrosine concentration c using the standard curve formula.

[0157] 3.2.3.4 Experimental data processing

[0158] Keratinase activity was expressed as X i The unit is enzyme activity unit per milliliter (U / mL), calculated according to the following formula:

[0159]

[0160] Where:

[0161] c——Concentration of L-tyrosine in the sample tube, in micrograms per milliliter (μg / mL);

[0162] c0——the concentration of L-tyrosine in the blank tube, in micrograms per milliliter (μg / mL);

[0163] N——dilution multiple of the sample;

[0164] 4——Total volume of enzyme reaction system, in milliliters (mL);

[0165] m——the amount of sample sucked (here 1mL), in milliliters (mL);

[0166] 10——Reaction time, in minutes (min).

[0167] The calculation result is rounded to an integer. The test result is based on the arithmetic mean of the parallel determination results.

[0168] Example 4 Basic properties of the produced keratinase

[0169] The keratinase mutant provided by the present invention has an optimum reaction pH of 8.5. After being treated at pH 6.5-12.5 for 20 hours, the relative enzyme activity of the mutated keratinase is still maintained at more than 80%; the optimum reaction temperature is 55° C., and the enzyme activity can still be retained at more than 90% under the condition of 40-75° C. for 20 hours, as follows:

[0170] (1) Optimal reaction pH

[0171] The supernatant obtained by centrifuging the fermentation broth of batch 3 in Example 2.1 and batch 1 in Example 2.2 was taken as the crude enzyme solution, and the keratinase activity was measured at pH values ​​of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, and 12.0 at a temperature of 40°C, and the relative enzyme activity was calculated, with the highest enzyme activity being 100%. The measurement results are as follows: Figure 2 As shown, the activity of the mutant keratinase is highest at pH 8.5.

[0172] (2) Alkali resistance

[0173] The supernatant obtained by centrifuging the fermentation broth of batch 3 in Example 2.1 and batch 1 in Example 2.2 was taken as a crude enzyme solution, and the pH of the enzyme solution was adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, and 13 with 0.1 M NaOH or 0.1 M HCl, respectively. After standing at room temperature for 20 hours, the enzyme activity was measured at 40° C. and pH 8.0, and the relative enzyme activity was calculated, with the original enzyme activity as 100%. The measurement results are as follows: Figure 3 As shown in the figure, after being treated at pH 7.0-9.5 for 20 hours, the relative enzyme activity of the mutant keratinase still remained above 90%, and the alkaline resistance was significantly improved compared with the wild-type keratinase.

[0174] (3) Optimum reaction temperature

[0175] The supernatant obtained by centrifugation of the fermentation broth of batch 3 in Example 2.1 and batch 1 in Example 2.2 was taken as the crude enzyme solution, and the keratinase activity was measured at 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95° C. under pH 8.0, and the relative enzyme activity was calculated, with the highest enzyme activity being 100%. The results are shown in FIG. Figure 4 As shown, the optimal reaction temperature of the mutated keratinase is 55°C.

[0176] (4) Thermal stability

[0177] The supernatant obtained by centrifuging the fermentation broth of batch 3 in Example 2.1 and batch 1 in Example 2.2 was taken as the crude enzyme solution, and the enzyme solution was placed in 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95°C for 20 hours. After the insulation, the enzyme activity was measured at 40°C and pH 8.0, and the relative enzyme activity was calculated, with the original enzyme activity as 100%. The experimental results are as follows: Figure 5 As shown in the figure, the mutant keratinase can still maintain more than 90% enzyme activity when kept at 40-75℃ for 20h. The thermal stability is significantly improved compared with the wild-type keratinase.

[0178] Example 5 Treatment of wool fabric with keratinase and determination of felt shrinkage

[0179] 100kg wool fabric was treated with 5000kg of water at 90℃ hot water for 5min, and then cooled to 55℃. The amount of keratinase in the experimental group (the enzyme solution of wild type and mutant keratinase was taken from the fermentation supernatant prepared in Example 2.1 and 2.3, respectively, and diluted to 100U / mL with boric acid buffer of pH 8.0) was 10kg, and no keratinase was added to the control group, only 10kg of water was added, and the treatment was carried out at 55℃, pH=8.5 for 20h. After treatment, the fabric was treated with 90℃ hot water to kill the enzyme for 5min, and after being fully washed, the fabric was conditioned for 12h at 25℃ and 65% relative humidity, and then the performance test of the felt shrinkage was carried out. According to the anti-felting standard (IWS TM No.31), the wool fabric was tested for the felt shrinkage using the Y(B)089A fully automatic shrinkage tester (produced by Wenzhou Darong Company) and the 5A program was selected.

[0180] It was determined that the felt shrinkage rate of the wool fabric in the control group was 11%, the felt shrinkage rate after treatment with the wild-type keratinase was 9%, and the felt shrinkage rate after treatment with the mutant keratinase was 6%.

[0181] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A keratinase mutant, characterized in that: The amino acid sequence of the keratinase mutant is shown in SEQ ID NO.

2.

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

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

4. A recombinant vector or recombinant strain comprising the coding gene according to claim 2.

5. The recombinant vector according to claim 4, characterized in that The expression plasmid used was pKS1.

6. The recombinant strain according to claim 4, characterized in that The host bacteria used was Bacillus amyloliquefaciens ZD-708 ( ).

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

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

9. The use of the keratinase mutant according to claim 1, characterized in that: It is used in the anti-felting treatment of wool fabrics.

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