Preparation method of high-activity keratinase and application of high-activity keratinase in fields of feather meal degradation, fermented feed preparation and like

Abstract

The invention belongs to the field of biotechnology and fermented feed, and relates to a preparation method of high-activity keratinase and application of the high-activity keratinase in the fields of feather meal degradation, fermented feed preparation and the like. The method comprises the following steps: constructing recombinant keratinase engineering bacteria and preparing crude enzyme liquid; the method comprises the following steps: carrying out eq-PCR (Polymerase Chain Reaction) amplification to obtain a kerD21eq fragment with a random mutation site, constructing an E.coli-B. subtilis shuttle expression vector plasmid library, and carrying out co-transformation on B.subtilis WB800N to obtain a mutant library; meanwhile, a high-throughput screening method combining a flat transparent ring and a 96-well plate method is established, high-activity keratinase is prepared on the basis of the screened mutant strain, degradation of feather meal is completed through the keratinase, and the keratinase can be applied to preparation of fermented feed. The feather waste is effectively utilized, remarkable economic benefits are brought, pollutants are effectively reduced, and the method has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and environmental management, and specifically relates to a preparation method of a high-activity keratinase and its application in the fields of feather powder degradation and fermented feed preparation. Background Art

[0002] As a major aquaculture country, my country has a huge demand for feed, and most of the feed protein resources, including soybean meal and fish meal, rely on imports. The prices of imported soybean meal and fish meal are high and unstable, which directly leads to an increase in feed costs and is easily affected by the international environment. This has greatly restricted the development of my country's feed industry and aquaculture. Therefore, it is particularly important to find some substitutes that meet domestic production capacity. Feather keratin is widely available and inexpensive, and is a protein resource with great potential. However, due to the structural characteristics of keratin, animals have a low digestibility and poor palatability for feather keratin. At present, feather meal cannot be used on a large scale in the aquaculture industry, and the current keratin processing methods are accompanied by serious pollution and nutrient loss.

[0003] Keratinase is an enzyme that can specifically degrade keratin and can effectively promote the degradation and conversion of feather keratin. However, naturally occurring keratinase is often accompanied by low activity, low yield, poor acid resistance and other problems, which cannot meet the needs of the industry. Therefore, the modification of existing wild-type keratinase has become an effective way to solve the urgent needs of the industry.

[0004] Directed evolution is an effective method for customizing biological proteins. Directed evolution refers to artificial simulation of natural evolution in a laboratory environment, and the selection of traits is completed by creating environmental pressure. The key link of directed evolution is random mutation. Among the various random mutation methods including physical and chemical mutagenesis, error-prone PCR, in vitro DNA shuffling, staggered extension, etc., error-prone PCR technology is the most commonly used as one of the safest, most reliable and efficient methods. A large number of mutants obtained by error-prone PCR need to be screened to obtain target mutants that meet the requirements; therefore, the establishment of high-throughput screening schemes is particularly important nowadays. Relying on high-throughput screening schemes, mutant screening can be completed in a shorter time. Therefore, combining error-prone PCR and high-throughput screening can help to complete directed evolution work quickly and efficiently. Summary of the invention

[0005] In view of the urgent need for high-activity keratinase in the current industry and the shortcomings of directed evolution screening methods, the present invention provides a method for preparing high-activity keratinase for feed and its application in the fields of feather degradation, feather powder fermented feed, etc. The present invention first constructs a vector and a recombinant engineered bacteria containing the vector, and prepares a crude enzyme solution based on the recombinant keratinase engineered bacteria; then eq-PCR amplification is used to obtain kerD21 with random mutation sites. eqfragments, constructed a plasmid library with E. coli-B. subtilis shuttle expression vector, obtained a mutant library by co-transformation of B. subtilis WB800N, and established a high-throughput screening method combining the flat plate transparent circle method and the 96-well plate method. The mutant strains were obtained by this high-throughput screening method, and a highly active keratinase was prepared; the degradation of feather powder substrate was completed by the enzyme, and it was applied in the actual feather powder fermentation feed environment; feather waste was further effectively utilized, which brought significant economic benefits and effectively reduced the generation of pollutants, and had good application prospects.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0007] The present invention provides a vector, comprising a vector A, a vector B and a vector C; wherein the vector A is obtained by connecting kerD21 and pHT-01-L, and is denoted as pHT-01-D21, wherein the nucleotide sequence of kerD21 is shown in SEQ ID NO.1; the vector B is obtained by connecting plasmid pHT-01-D21 and a signal peptide; the signal peptide is SP Q7 , whose encoding is SP Q7 The gene nucleotide sequence is shown in SEQ ID NO.5; vector C is obtained by connecting pHT-01-KerD21124D-124N 126A-126E with a signal peptide; the nucleotide sequence of the gene encoding pHT-01-KerD21124D-124N 126A-126E is shown in SEQ ID NO.3, and the signal peptide is SP Q7 , whose encoding is SP Q7 The nucleotide sequence of the gene is shown in SEQ ID NO.5.

[0008] Furthermore, a recombinant engineering bacterium carrying the vector is provided.

[0009] The present invention also provides a method for preparing a highly active keratinase, wherein the highly active keratinase is obtained by inoculating the recombinant engineered bacteria into a fermentation medium, culturing the medium, and then collecting the supernatant by centrifugation; wherein the medium is an LB medium, and the culture conditions are 37°C, 200rpm, and culturing for 12-14h.

[0010] The preparation method of the highly active keratinase specifically comprises the following steps:

[0011] 1. The keratinase gene of Pseudomonas aeruginosa PAD21 (GeneBank CP012582.1) was selected and codon optimized using Bacillus subtilis as a host. The codon-optimized recombinant keratinase gene was recorded as kerD21, and its nucleotide sequence was as SEQ ID NO.1, and its encoded amino acid sequence was as SEQ ID NO.2;

[0012] 2. The pHT-01 vector was double-digested with restriction endonucleases to obtain a linearized vector pHT-01-L; the kerD21 gene described in step 1 was used as a template for PCR amplification to obtain kerD21 containing restriction sites SpeI and SacII, and then kerD21 was connected to pHT-01-L and transformed into Escherichia coli (E. coli DH5α) recipient bacteria, and the recombinant shuttle plasmid was obtained by extracting the plasmid and recorded as pHT-01-D21. Finally, pHT-01-D21 was transformed into Bacillus subtilis WB800N to obtain an engineered bacterium that can be used for exocrine expression and production of keratinase KerD21, recorded as B. subtilis WB800N-pHT-01-D21;

[0013] 3. The recombinant keratinase engineered bacteria B. subtilis WB800N-pHT-01-D21 obtained in step 2 was inoculated into LB medium, and after culturing for a period of time, the fermentation liquid obtained after the cultivation was centrifuged to separate the bacteria, and the obtained supernatant was recorded as KerD21;

[0014] Preferably, in step 3, the inoculation amount of the recombinant keratinase engineering bacteria inoculated into the LB culture medium is 1% (g / mL); the culture conditions are 37° C., 200 rpm, and 12-14 h.

[0015] 4. Using the kerD21 gene described in step 1 as a template, error-prone PCR (eq-PCR) amplification was performed to obtain the target fragment kerD21 eq , and kerD21 eq kerD21 was obtained by double restriction digestion eq-c kerD21 was ligated using T4 DNA ligase eq-c Connect to the linearized fragment of the expression vector pHT-01-L, and then transform the E. coli (E. coli DH5α) recipient bacteria. After transformation, the recombinant shuttle plasmid library is obtained by co-extraction, which is recorded as pHT-01-D21 RM Finally, pHT-01-D21 RMCo-transformed Bacillus subtilis WB800N to obtain a recombinant keratinase mutant strain library that can be used for secretory expression screening, denoted as B. subtilis WB800NpHT-01-D21 RM ;

[0016] 5. Establishment of a high-throughput screening method for keratinase KerD21 mutants:

[0017] First, a milk screening medium is prepared. The milk screening plate is prepared by adding 1-1.5% skimmed milk powder to LB solid medium, and controlling the content of peptone to be no more than 6%. A milk screening plate is prepared based on the milk screening medium, and then the B. subtilis WB800N-pHT-01-D21 obtained in step 4 is added to the plate. RM The transformants were spread on milk screening plates. After culture, the sizes of the transparent zones were compared, and the ratio of the transparent zone to the colony area was calculated. The mutant with the highest ratio was recorded as KerD21 M, and the corresponding recombinant engineered bacteria was recorded as B. subtilis WB800NpHT-01-D21-M.

[0018] Preferably, the culture conditions in step 5 are: 37° C., 200 rpm, 10-12 h; the milk screening plate; contains components by mass fraction: skim milk powder 1.5%, peptone 5%, yeast extract powder 5%, NaCl 10%, agar 10-15%;

[0019] 6. First, the recombinant engineered bacteria B. subtilis WB800N pHT-01-D21-M was inoculated into a 96-well plate containing LB liquid culture medium, and the 96-well plate was sealed with a sealing film to culture the bacteria to obtain a cultured bacterial solution;

[0020] The cultured bacterial liquid was inoculated into a 96-well deep-well plate containing LB medium for cultivation, and the supernatant was collected by centrifugation after cultivation;

[0021] The supernatant was added to a 96-well deep-well plate, and feather powder and Tris-HCl (pH=7.0) buffer were added for mixed reaction. After the reaction, trichloroacetic acid was added to terminate the reaction. The supernatant was collected by centrifugation and the OD of the supernatant was measured. 280nm The mutant with the highest absorption value was recorded as KerD21124D-124N 126A-126E, and the corresponding expression vector was recorded as pHT-01-KerD21124D-124N 126A-126E. The nucleotide sequence of its encoding gene is shown in SEQ ID NO.3; and the amino acid sequence is shown in SEQ ID NO.4;

[0022] Preferably, the conditions for bacterial culture in step 6 are: placing in a constant temperature shaking shaker at 37°C and 180rpm for overnight culture; the inoculation amount of the cultured bacterial solution inoculated into the LB medium is 1%; the culture conditions are 37°C and 200rpm for 10-14h; the centrifugation conditions are: centrifugation at 4000rpm for 15min;

[0023] The reaction time is 1 hour, and the temperature is 37-40° C.; the dosage of the feather powder, the supernatant, the Tris-HCl buffer and the trichloroacetic acid is in the following relationship: 0.0094 g: 0.2 mL: 0.4 mL: 0.4 mL, wherein the pH of the Tris-HCl buffer is 7.0, the concentration is 0.05 mol / L, and the concentration of the trichloroacetic acid is 10%.

[0024] 7. Based on the pHT-01-D21 obtained in step 2, the signal peptide was connected to pHT-01-D21 to construct a pHT-01-D21-SP vector, and the Escherichia coli (E. coli DH5α) recipient bacteria were transformed; the recombinant signal peptide shuttle plasmid was obtained by extraction, and then the recombinant signal peptide shuttle plasmid was transformed into Bacillus subtilis WB800N to obtain a recombinant keratinase signal peptide library that can be used for secretory expression screening, recorded as B. subtilis WB800NpHT-01-D21-SP;

[0025] 8. Follow the steps in step 3, except that B. subtilis WB800N pHT-01-D21-SP is replaced with B. subtilis WB800N-pHT-01-D21; the resulting supernatant is designated as KerD21-SPS;

[0026] KerD21-SPS was mixed with Tris-HCl buffer and feather powder for reaction. After the reaction, trichloroacetic acid was added to terminate the reaction and the OD was measured. 280nm The best mutant was selected as the best signal peptide, and the best signal peptide was SP Q7 ; Its code SP Q7 The nucleotide sequence of the gene is shown in SEQ ID NO.5;

[0027] Preferably, the reaction time in step 8 is 1 h, and the temperature is 37-40° C.; the dosage of KerD21-SPS, Tris-HCl buffer, feather powder and trichloroacetic acid is in the following relationship: 1 mL: 2 mL: 0.047 g: 2 mL; the pH of Tris-HCl buffer is 7.0, the concentration is 0.05 mol / L, and the concentration of trichloroacetic acid is 10%.

[0028] 9. Use double enzyme digestion method to ligate SP Q7Connect with the expression vector pHT-01-KerD21124D-124N126A-126E obtained in step 6 to obtain a vector containing a highly efficient signal peptide SP Q7 The KerD21124D-124N 126A-126E mutant expression vector was labeled pHT-01-KerD21124D-124N 126A-126E (SP Q7 ); and pHT-01-KerD21124D-124N126A-126E (SP Q7 ) was transformed into Bacillus subtilis WB800N to obtain a recombinant engineered bacterium, which was recorded as B. subtilis WB800N-pHT-01-KerD21124D-124N 126A-126E (SP Q7 );

[0029] Finally, follow the steps in step 3. The difference is that B. subtilis WB800N-pHT-01-KerD21124D-124N126A-126E (SP Q7 ) was replaced with B. subtilis WB800N-pHT-01-D21; the resulting supernatant was a highly active keratinase, denoted as KerD21124D-124N 126A-126E (SP Q7 ).

[0030] The SP Q7 It is a highly efficient signal peptide. Compared with its wild-type signal peptide and other types of signal peptides, it has higher hydrophobicity and higher α-helix content. It can enhance the expression efficiency of the SEC secretion pathway of Bacillus subtilis and improve the effective activity.

[0031] Preferably, the Bacillus subtilis WB800N described in the step refers to Bacillus subtilis in which eight protease genes, AprE, NprE, NprB, Vpr, Epr, Bpr, Mpr, and WprA, are knocked out, and is named Bacillus subtilis WB800N (B. subtilis WB800N), which can effectively improve the success rate of secretory expression of recombinant exogenous proteins and the amount of protein secretion.

[0032] Based on the use of high-activity keratinase for feather meal degradation, the operation is as follows:

[0033] Feather meal is used as raw material, crude keratinase enzyme solution is added, Mg 2+forming a mixed system; controlling the pH of the mixed system, and reacting under certain temperature conditions to degrade keratin in feathers; the crude keratinase solution includes KerD21, KerD21124D-124N 126A-126E and KerD21124D-124N 126A-126E (SP Q7 ).

[0034] Preferably, the amount of crude keratinase solution added is 1%-25% of the mixed system based on the volume fraction of the mixed system. 2+ The concentration of Mg is 1-5mM; the reaction temperature is 37℃ and the reaction time is 1h; 2+ Including MgCl2.

[0035] Based on the use of high-activity feed keratinase for preparing feather powder fermented feed, the steps are as follows

[0036] First, feather meal, bean dregs and second-rate flour are mixed to obtain a mixed raw material; then fermented seed liquid of saccharomyces cerevisiae, bacillus licheniformis, lactobacillus fermentation and lactobacillus rhamnosus are inoculated, crude keratinase enzyme liquid, methionine (Met), lysine (Lys) and mineral ions are added, and finally water is added to the target moisture content, and anaerobic fermentation is performed, and fermented feed is obtained after the fermentation is completed; the crude keratinase enzyme liquid includes KerD21, KerD21124D-124N 126A-126E and KerD21124D-124N126A-126E (SP Q7 ).

[0037] Preferably, the mass ratio of feather meal, bean dregs and inferior powder is 5:3:2, the total inoculation amount of fermentation bacteria liquid of Saccharomyces cerevisiae, Bacillus licheniformis, Lactobacillus fermentum and Lactobacillus rhamnosus is 6% of the mass of the mixed raw materials (v / m, mL / g; that is, the total inoculation amount of the fermentation bacteria liquid and the amount of the mixed raw materials are 6mL:100g), wherein the volume ratio of the fermentation seed liquid of Saccharomyces cerevisiae, Bacillus licheniformis, Lactobacillus fermentum and Lactobacillus rhamnosus is 2:2:1:1;

[0038] The mineral ions include CaCO3, which is added in an amount of 2% of the mass of the mixed raw materials. At the same time, 0.20-0.30% of the mass of the mixed raw materials is added, and the addition amount of Met (methionine) is added in an amount of 0.20-0.30% of the mass of the mixed raw materials, and the addition amount of Lys (lysine) is added in an amount of 0.25% of the mass of the mixed raw materials.

[0039] The amount of crude keratinase solution added is 1%-15% of the mass of the mixed raw materials (v / m, mL / g; that is, the amount of crude keratinase solution to the mixed raw materials is 1-15mL: 100g); the target moisture content is 35-45%; the fermentation temperature is 35-40°C, and the fermentation time is 7 days.

[0040] The advantages and technical effects of the present invention are:

[0041] (1) The present invention provides a method for producing keratinase, which can quickly and efficiently obtain a large amount of keratinase through a Bacillus subtilis secretion expression system, realize the industrial production of keratinase, and obtain a keratinase product with good stability and enzyme activity.

[0042] (2) The present invention provides a keratinase directed evolution technology solution, and establishes a high-throughput screening method based on 96-well plates and a flat plate transparent circle method. Compared with other traditional high-throughput screening methods, the flat plate transparent circle method can quickly and efficiently complete the initial screening and effectively select positive mutants; the 96-well plate is used to complete the rescreening, which can quantify the activity, realize the connection between the enzyme and the substrate molecule it transforms, and the connection between the genotype and the phenotype, so as to quickly measure and screen for keratinase with high catalytic activity. By introducing random mutation sites through eq-PCR, establishing a mutant library by relying on a secretory expression system, the flat plate transparent circle method and 96-well plate high-throughput screening, the improved production of keratinase is achieved, and a keratinase product with high catalytic activity is obtained.

[0043] (3) The crude keratinase solution prepared by the present invention can be used for the degradation of keratin. Pretreatment experiments on feather powder show that keratinase can significantly improve the degradation of feather keratin.

[0044] (4) The method for preparing the feather powder fermented feed provided by the present invention has low cost and simple process, and can solve the technical problem that feather waste cannot be effectively utilized; in addition, keratinase has high activity and strong stress resistance, and has broad industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the original sequence of the keratinase gene.

[0046] Figure 2 This is the electrophoresis diagram of the PCR amplified fragment of keratinase gene.

[0047] Figure 3 This is the physical map of the recombinant shuttle plasmid pHT-01-D21 used for secretory expression in Bacillus subtilis.

[0048] Figure 4 This is a Western-Blot verification diagram of the secretory expression of keratinase KerD21 by the recombinant engineered bacteria B. subtilis WB800N pHT-01-D21.

[0049] Figure 5 is the relative enzyme activity of recombinant keratinase.

[0050] Figure 6 This is the electrophoresis diagram of the eq-PCR amplified fragment of the keratinase gene kerD21.

[0051] Figure 7 Recombinant shuttle expression vectors pHT-01-D21 and kerD21 eq Double restriction enzyme digestion electrophoresis of mutant gene.

[0052] Figure 8 The recombinant engineering bacteria B. subtilis WB800N pHT-01-D21 RM Electrophoresis diagram of PCR verification of some positive transformants in the mutant library.

[0053] Fig. 9 This is a degradation verification diagram of the flat plate transparent circle method.

[0054] Fig.10 It is the nucleotide sequence of keratinase mutant KerD21124D-124N 126A-126E.

[0055] Fig.11 Comparison of the nucleotide sequences of keratinase mutant KerD21124D-124N 126A-126E and wild-type keratinase Ker D21. DETAILED DESCRIPTION

[0056] The present invention is further defined in the following examples. Based on the above description and these examples, those skilled in the art can determine the essential characteristics of the present invention and can make various modifications to the present invention without departing from the spirit and scope of the present invention to make it suitable for various uses and conditions.

[0057] Reagent Material Description:

[0058] Feather meal was purchased from Shandong Yuduobao Company;

[0059] The recipient bacteria E. coli DH5α was a chemical transformation competent cell prepared using the E. coli DH5α strain, purchased from Nanjing Novozymes Biotech Co., Ltd.; Tris-HCl and trichloroacetic acid were purchased from Shanghai Sinopharm;

[0060] LB medium is a common microbial medium suitable for the growth and expression of Bacillus subtilis, with a formula of 10% tryptone, 5% yeast extract powder, and 10% sodium chloride;

[0061] Bacillus subtilis WB800N refers to Bacillus subtilis with eight protease genes, AprE, NprE, NprB, Vpr, Epr, Bpr, Mpr, and WprA, knocked out, named B.subtilisWB800N; it can effectively improve the secretion success rate of recombinant exogenous proteins and the amount of secreted protein expression. As a Bacillus subtilis receptor strain, it is commonly used in various academic studies related to heterologous expression of Bacillus subtilis;

[0062] Among them, the B. subtilisWB800N competent state preparation method refers to the literature (Zhang Tianxi. Expression and identification of thermostable esterase of Clostridium thermocellum and surface display of Bacillus subtilis spores, [Master's thesis]. Zhenjiang, Jiangsu: Jiangsu University, 2016.).

[0063] The shuttle expression vector pHT-01 used was purchased from Beijing Biosai Biotechnology Co., Ltd.;

[0064] In the preparation of feather meal feed, the fermentation strains used were Saccharomyces cerevisiae (CGMCC 2.1527), Bacillus licheniformis (CGMCC 1.813), Lactobacillus fermentum (CGMCC 1.15608) and Lactobacillus rhamnosus (CGMCC 1.577), all of which were purchased from the China General Microbiological Culture Collection Administration Center. The yeast was inoculated into LB medium at a 1% inoculum and cultured at 30°C and 150 rpm in a shaking incubator until the OD 600nm = 0.8-1.0; the spore-forming bacteria were inoculated into LB medium at 1% inoculum at 37°C and shaken at 150 rpm until OD 600nm =0.8-1.0. Lactic acid bacteria were inoculated into MRS medium at 1% inoculum and cultured anaerobically at 37°C until OD 600nm =0.8-1.0; the corresponding fermentation seed solution is obtained. The inoculation amount of 1% means that 1g of bacteria is inoculated per 100mL of culture medium.

[0065] During the evaluation of the fermentation effect of feed, the determination method of crude protein and small peptide content refers to the national standard GB / T22492-2008; the free amino acid determination method refers to the Henan Provincial Standard DB41 / T 1727-2018, among which the extraction step of free amino acids in feed samples refers to the acid extraction method in the national standard GB / T 18246-2019.

[0066] In the following embodiment, a method for determining the activity of keratinase using feather powder as a substrate is involved. The specific operation is as follows: 1.0 mL of keratinase (engineered bacteria culture supernatant) is taken, 2.0 mL of the corresponding pH buffer (sodium hydrogen citrate-citric acid buffer (phosphate buffer) is used when pH 3-7, Tris-HCl buffer is used when pH 7-9, and Gly-NaOH buffer is used when pH 9-11) is added, 0.047 g of feather powder is added as a substrate, and the reaction is carried out in a constant temperature shaker at 30°C or 40°C. After 1 hour, 2.0 mL of 10% trichloroacetic acid (TCA) is added to terminate the reaction, and the supernatant is centrifuged at 10000 rpm and 4°C for 10 minutes, and the OD value of the supernatant is measured. 280nm The absorbance of the reaction tubes was 0.01, and the reaction tubes treated with TCA before the reaction were used as controls. Enzyme activity was defined as 1 enzyme activity unit (U) when the absorbance at 280 nm increased by 0.01 under the above reaction conditions.

[0067] The ELISA plate is a 96-well microplate that can be quickly scanned and measured using an ELISA reader. The 96-well deep-well plate is a large-volume 96-well plate used for batch induction expression and reaction.

[0068] Example 1: Preparation method of crude recombinant keratinase enzyme solution;

[0069] Construction of recombinant keratinase engineering bacteria:

[0070] (1) The keratinase gene sequence of Pseudomonas aeruginosa PAD21 in NCBI (Gene Bank CP012582.1) was optimized according to the codon preference in Bacillus subtilis to obtain kerD21. Figure 1 is the original sequence of the keratinase kerD21 gene, and the nucleotide sequence of the codon-optimized kerD21 gene is shown in SEQ ID NO.1.

[0071] Primers were designed to amplify the target gene sequence. The forward primer sequence was shown in SEQ ID NO.6, and the reverse primer sequence was shown in SEQ ID NO.7. The electrophoresis diagram of the PCR amplified target gene fragment was shown in Figure 2 shown.

[0072] (2) Recombinant keratinase engineering bacteria:

[0073] S1. Double-digest the pHT-01 vector with restriction endonucleases to obtain the linearized vector pHT-01-L. Use the kerD21 gene described in step 1 as a template to perform PCR amplification to obtain kerD21 containing restriction sites SpeI and SacII, gel-recover the linearized target fragment kerD21-C, and measure the product concentration. Use T4 DNALigase to connect the double-digested linearized plasmid vector pHT-01-L and the kerD21-C gene fragment, respectively. Transform the ligation products into E.coli DH5α competent cells, pick the positive transformants to extract the recombinant plasmid, and obtain the recombinant shuttle plasmid pHT-01-D21. The map is shown in Figure 3 shown.

[0074] S2, transform the recombinant shuttle plasmid pHT-01-D21 obtained above into B. subtilis WB800N competent cells, spread on LB plates and culture overnight (37°C), then test the positive transformants by colony PCR to verify that the correct strain is the recombinant keratinase engineering bacteria B. subtilis WB800NpHT-01-D21;

[0075] The recombinant shuttle expression vector pHT-01 was treated according to step S2, that is, pHT-01-D21 was replaced with pHT-01 to obtain the recombinant engineered bacteria B. subtilis WB800NpHT-01, which was used as a blank control.

[0076] (3) Expression identification of recombinant engineered bacteria B. subtilis WB800NpHT-01-D21:

[0077] The recombinant engineered bacteria B. subtilis WB800NpHT-01-D21 was inoculated into LB culture medium at an inoculum amount of 1% (m / v, g / mL, i.e., 1 g of the recombinant engineered bacteria was inoculated per 100 mL of culture medium) and cultured at 37°C and 200 rpm for 12 hours. After the culture was completed, the fermentation broth was collected and centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected to obtain keratinase KerD21.

[0078] The extracted proteins were verified by Western Blot analysis. The primary antibody used was 6X histag antibody with a concentration of 1:2000, and the secondary antibody was D110087HRP-labeled goat anti-mouse IgG with a concentration of 1:5000. The Omni-ECLTM Basic Chemiluminescence Kit (YaZyme) was used for color development. Figure 4As shown, a single specific band can be seen near 33 kDa for the secreted protein in the fermentation broth of the recombinant engineering bacteria B. subtilis WB800N pHT-01-D21, while no band appeared in the fermentation broth of the control recombinant engineering bacteria B. subtilis WB800N pHT-01, indicating that the recombinant keratinase KerD21 was successfully expressed in Bacillus subtilis.

[0079] Activity determination and property analysis of recombinant keratinase:

[0080] The activity of keratinase KerD21 was measured with feather meal as substrate, and the reaction pH range was set to 3.0-7.0, and each change of 1 pH was used as a measurement interval; the substrate was feather meal, the addition amount was 0.047g, the reaction system was 5mL, 0.05M Tris-HCl (pH=7.0) buffer was added 2mL, the reaction conditions were shaker 180rpm, reaction temperature 37℃, reaction time 1h, and 2mL, 10% trichloroacetic acid was added to terminate the reaction. The relative enzyme activity of recombinant keratinase is as follows Figure 5 As shown, it was determined that KerD21 can maintain 35% of its maximum activity at pH 4-5. This proves that keratinase KerD21 has a certain acid resistance and has a wider range of applications compared to other wild-type alkaline keratinase, filling the blank area of ​​acidic keratinase.

[0081] (4) Directed evolution and high-throughput screening of recombinant keratinase;

[0082] Construction of recombinant engineered bacteria B. subtilis WB800NpHT-01-D21 RM Mutation library:

[0083] Using the kerD21 gene as a template, according to the user manual of the adjustable eq-PCR kit, error-prone PCR amplification was performed using two end primers containing restriction sites Spe I and Sac II. The forward primer sequence is shown in SEQ ID NO.6, and the reverse primer sequence is shown in SEQ ID NO.7. The eq-PCR reaction system is: 94°C, 3 min, 98°C, 10 s, 60°C, 15 s, 72°C, 30 s, 30 cycles; the linearized fragment of the target gene amplified by eq-PCR is recorded as kerD21 eq ,like Figure 6 As shown; and kerD21 eq kerD21 was obtained by double restriction digestion eq-c .

[0084] The size of the digested product was verified by 1% agarose gel electrophoresis. Figure 7 As shown, kerD21 eq-cThe mutant gene fragment had an obvious band at 900 bp, and the plasmid vector had obvious bands at about 8000 bp and 900 bp, respectively. The linearized vector and the target mutant fragment were recovered by gel and the product concentration was determined.

[0085] The shuttle expression vector pHT-01 was double-digested with restriction endonucleases Spe I and Sac II to obtain pHT-01-L. T4 DNA Ligase was used to linearize the double-digested plasmid vector pHT-01-L and kerD21 eq-c The gene fragments were connected, and the connected products were co-transformed into E. coli DH5α competent cells, coated on solid LB medium plates and cultured at 37°C for 12 hours. Translucent colonies were observed on the plates, and some of the transformants were picked for PCR verification as positive transformants. At the same time, all transformants were eluted with 5 mL of LB liquid medium from each plate, and then transferred to LB liquid medium and cultured until OD 600nm = 0.6-0.8, and co-extract the plasmid to obtain the mutant plasmid library pHT-01-D21 RM .

[0086] Then the mutant gene library plasmid pHT-01-D21 RM Transform B. subtilis WB800N competent cells, spread on LB solid medium plates and culture for 12 hours, pick transformants and verify the positive transformants by PCR; 1% agarose gel electrophoresis is used to detect transformants, such as Figure 8 As shown, a clear band appeared at 1500 bp, proving that the transformation was successful. That is, a recombinant keratinase mutant strain library that can be used for secretory expression screening was obtained, B. subtilis WB800N pHT-01-D21 RM .

[0087] (5) Establishment of high-throughput screening method:

[0088] Milk screening plate: Keratinase is a protease that can degrade keratin specifically and some other types of proteins. Based on this feature, this experiment uses skim milk as a substrate and adds it to LB solid medium to make a milk screening medium. The mass fractions of the components of the milk screening medium are: 1.5% skim milk powder, 5% peptone, 5% yeast extract powder, and 10% NaCl.

[0089] Prepare milk screening plates based on milk screening medium and then add B. subtilis WB800N pHT-01-D21 RM The engineered bacteria and the blank control of B. subtilis WB800NpHT-01 were diluted and spread on the plate, cultured at 37℃ for 12h, and the transparent zone was verified. Fig. 9 As shown in the figure, a degradation transparent circle appeared in the engineered bacteria plate (a), while no transparent circle was seen in the blank control (b), proving that B. subtilis WB800NpHT-01 engineered bacteria could not effectively degrade milk, while B. subtilis WB800N pHT-01-D21 RM Keratinase can be produced and is able to degrade efficiently, so this method can be used for screening.

[0090] The ratio of the transparent zone to the colony area was further calculated, and the mutant with the largest ratio was selected and recorded as KerD21 M; and the colony (recombinant engineered bacteria) corresponding to the largest ratio was recorded as B. subtilis WB800NpHT-01-D21-M;

[0091] (6) In a sterile clean bench, pick the monoclonal transformants of the recombinant engineered bacteria B. subtilis WB800N pHT-01-D21-M mutant library one by one into a 96-well plate containing 100 μL LB liquid medium in each well, seal the 96-well plate with a sealing film, place it in a constant temperature shaking incubator at 37°C and 180 rpm for 12 h, and take the bacterial solution cultured overnight in the 96-well plate;

[0092] Then, the bacterial liquid was transferred to a 96-well deep-well plate containing LB medium (500 μL LB medium / well) at a volume ratio of 1%, sealed with a rubber cover, and placed in a constant temperature shaking shaker at 37°C and 180 rpm for 12 h; after the culture was completed, the supernatant was collected by centrifugation at 4000 rpm for 15 min using a 2*2*96 Holes desktop low-speed centrifuge;

[0093] Add 0.0094g feather powder, 0.2mL supernatant and 0.4mL Tris-HCl buffer solution to each well of the deep-well plate, where the Tris-HCl buffer solution has a pH of 7.0 and a concentration of 0.05mol / L. Place in a 37°C constant temperature shaking incubator for 1 hour. After the reaction is completed, add 0.4mL of 10% trichloroacetic acid to terminate the reaction; centrifuge at 4000rpm for 15 minutes using a 2*2*96Holes desktop low-speed centrifuge to collect the supernatant, dilute the supernatant 5 times with ddH2O, and transfer 100μL to a quartz ELISA plate to measure OD 280nm Absorbance, OD 280nm The higher the absorbance value, the higher the activity of the mutant. Using the non-mutant strain as a control, screen the mutant strain with obvious degradation;

[0094] In the first round of screening on milk screening plates, 3000 transformants were sequentially picked from the mutant plates by the plate clearing zone method;

[0095] In the second round of screening, four positive mutants with outstanding activity were screened by 96-well plate method, which were respectively recorded as KerD21T1-1 (abbreviated as T1-1), KerD21 T1-2 (abbreviated as T1-2), KerD21 T1.2-2 (abbreviated as T1.2-2), and KerD21124D-124N126A-126E (abbreviated as T2.2-4). The four colonies were measured by keratinase activity assay method to determine their activity. The enzyme activity of the unmutated B. subtilis WB800NpHT-01-D21 was used as the control group and recorded as 100%. The relative enzyme activity of each mutant was calculated, among which the group with the highest enzyme activity was KerD21124D-124N 126A-126E, with the highest being 180.17±4.24%.

[0096] Table 1. Comparison of activities of KerD21 mutants

[0097]

[0098] The mutants were sent to Shanghai Biotechnology Co., Ltd. for sequencing. Fig.10 is the nucleotide sequence of keratinase mutant KerD21124D-124N 126A-126E; Fig.11 The nucleotide sequence of the keratinase mutant KerD21124D-124N 126A-126E is compared with that of the wild-type keratinase Ker D21. The red marks in the figure represent the nucleotide mutation sites. Finally, the recombinant keratinase engineering bacteria B.subtilis WB800N pHT-01-KerD21124D-124N126A-126E with high catalytic activity was obtained. The keratin analog AAPF was used as a substrate, and the substrate docking simulation was performed on it. Then the lowest energy conformation was taken, and the analysis showed that the substrate binding energy of the mutant KerD21124D-124N 126A-126E was reduced from -7.6kcal / mol to -8.4kcal / mol, and due to the mutation, two hydrogen bonds that did not exist originally can be formed between the active center of the enzyme and the substrate. The two hydrogen bonds are formed by tryptophan No. 115 and asparagine No. 163 with the substrate respectively. The addition of these two hydrogen bonds further enhances the binding between the enzyme and the substrate. Therefore, the improvement of the enzyme activity of KerD21124D-124N 126A-126E is in line with theory; it can be used to prepare high-activity keratinase for feed.

[0099] (7) Optimization of recombinant keratinase KerD21 signal peptide

[0100] Based on the pHT-01-D21 obtained in step 1, the signal peptide was connected to pHT-01-D21 by double enzyme digestion and enzyme ligation to construct a pHT-01-D21-SP vector, and the Escherichia coli (E. coli DH5α) recipient bacteria were transformed; a recombinant signal peptide shuttle plasmid was obtained by extraction, and the shuttle plasmid was used to transform Bacillus subtilis WB800N to obtain a recombinant keratinase signal peptide library that can be used for secretory expression screening, which was recorded as B. subtilis WB800N pHT-01-D21-SP; the signal peptides were selected as: SP wapA , SP NprE , SP DacB , SP AmyE , SP AprE , SP Q7 , SP LipA ;

[0101] (8) B. subtilis WB800NpHT-01-D21-SP was inoculated into 100 mL LB liquid medium at an inoculum amount of 1% of the volume of the medium and cultured at 37°C and 200 rpm for 12 h. After the culture was completed, the fermentation broth was collected and centrifuged at 8000 rpm for 10 min. The supernatant was collected and 1 mL of the obtained supernatant was taken. 2 mL of 0.05 M Tris-HCl (pH = 7.0) and 0.047 g of feather powder were added. The reaction was carried out at 37°C for 1 h. After the reaction was completed, 2 mL of 10% trichloroacetic acid was added to terminate the reaction. The supernatant was centrifuged at 10000 rpm for 10 min. The OD 280nm Absorption value, with high absorbance value as the standard of high activity; the recombinant engineering bacteria B.subtilis WB800N pHT-01-D21-SP was screened for exocrine expression, and a variety of signal peptides (SP wapA , SP NprE , SP DacB , SP AmyE , SPAprE, SP Q7 , SP LipA ) and KerD21 wild-type signal peptide, wherein the signal peptide with high catalytic activity is SP Q7 , whose encoding is SP Q7 The nucleotide sequence of the gene is shown in SEQ ID NO.5;

[0102] Table 2. Comparison of KerD21 signal peptide activity

[0103]

[0104] SP Q7Connect with the obtained expression vector pHT-01-KerD21124D-124N 126A-126E to obtain a highly efficient signal peptide SP Q7 The KerD21124D-124N 126A-126E mutant expression vector was labeled pHT-01-KerD21124D-124N 126A-126E (SP Q7 ); and pHT-01-KerD21124D-124N 126A-126E (SP Q7 ) was transformed into Bacillus subtilis WB800N to obtain a recombinant engineered bacterium, which was recorded as B. subtilis WB800N-pHT-01-KerD21124D-124N 126A-126E (SP Q7 );

[0105] Finally, B. subtilis WB800N-pHT-01-KerD21124D-124N 126A-126E (SP Q7 ) was inoculated into LB medium at an inoculum size of 1%, and cultured at 37°C and 200 rpm for 12 h; after induction culture, the fermentation broth obtained after the culture was completed was centrifuged to separate the bacteria, and the supernatant obtained was the highly active keratinase, which was recorded as KerD21124D-124N 126A-126E (SP Q7 ).

[0106] Embodiment 2:

[0107] According to KerD21, KerD21124D-124N 126A-126E and KerD21124D-124N 126A-126E (SP Q7 ) Prepare feather powder fermented feed, mainly puffed feather powder, supplemented with bean dregs and second-grade powder;

[0108] First, feather powder, bean dregs and second-rate powder are mixed in a mass ratio of 5:3:2 to obtain a mixed raw material; then, fermented bacterial strains of saccharomyces cerevisiae, bacillus licheniformis, lactobacillus fermentation and lactobacillus rhamnosus are inoculated, and the inoculation amount is 6% (m / v, g / mL) of the mass of the mixed feed, wherein the volume ratio of the fermented bacterial strains of saccharomyces cerevisiae, bacillus licheniformis, lactobacillus fermentation and lactobacillus rhamnosus is 2:2:1:1; then Met, Lys and mineral ions are added, and finally water is added to the target water content to form a reaction system, and after mixing, a one-way valve anaerobic fermentation bag is loaded, and the bag is placed in a constant temperature incubator for anaerobic fermentation, and fermented feed is obtained after the fermentation is completed. The mineral ion is CaCO3, and the addition amount is 2% of the mass of the mixed raw material, the addition amount of Met is 0.25% of the mass of the mixed raw material, and the addition amount of Lys is 0.25% of the mass of the mixed raw material.

[0109] Each package of feed contains 50g of mixed raw materials and is fermented in a sealed package using a one-step fermentation method; the fermentation temperature is 37°C and the fermentation time is seven days.

[0110] Four groups were set up to measure the pH and small peptide content of each group; the specific groups are as follows:

[0111] ① No fermentation group, that is, the reaction system was not fermented, which was recorded as unfermented;

[0112] ② The reaction system was fermented, but the fermentation bacteria were not inoculated, which was recorded as no fermentation bacteria were added;

[0113] ③ Adding 1% and 15% KerD21 to the mixed system for fermentation was recorded as 1% KerD21 and 15% KerD21 respectively;

[0114] ④ 1% and 15% of KerD21124D-124N 126A-126E were added to the mixed system for fermentation, which were recorded as 1% KerD21124D-124N 126A-126E and 15% KerD21124D-124N 126A-126E respectively;

[0115] ⑤ Add 1% and 15% of KerD21124D-124N 126A-126E (SP Q7 ) were fermented and recorded as 1% KerD21124D-124N 126A-126E (SP Q7 ) and 15% KerD21124D-124N 126A-126E (SP Q7 )

[0116] Determination of small peptide content in feather meal fermented feed: Weigh equal weight of fermented feed, dry at constant temperature and digest, and determine the protein small peptide content by Kjeldahl method;

[0117] Table 3. Small peptide content of fermented feed

[0118]

[0119]

[0120]

[0121] It can be seen that the crude keratinase solution prepared by the present invention can be used for the degradation of keratin, and significantly improves the degradation of feather keratin. At the same time, the present invention provides a method for preparing a feather powder fermented feed with low cost and simple process, which can solve the technical problem that feather waste cannot be effectively utilized; and the keratinase has high activity and strong stress resistance, and has a wide prospect of industrial application.

[0122] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, a person of ordinary skill in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

[0123] Sequence Listing

[0124] SEQ ID NO.1

[0125] CATATGGCGGAAGCGGGCGGTCCTGGCGGTAATCAGAAAATTGGCAAATATACATACGGTAGCGATTATGGCCCGCTGATTGTGAATGATCGCTGCGAAATGGATGATGGTAATGTGATTACCGTTGATATGAATGGCTCAACCAATGATTCAAAAACAACACCGTTTCGTTTTGCATGCCCGACCAATACATATAAACAGGTGAATGGCGCGTATTCACCGCTGAATGATGCGCATTTCTTTGGTGGCGTTGTTTTTAATCTGTATCGCGATTGGTTTGGTACAAGCCCGCTGACACATAAACTGTATATGAAAGTTCATTACGGCCGCTCAGTTGAAAATGCGTATTGGGATGGTACAGCGATGCTGTTTGGTGACGGCGCGACAATGTTTTATCCGCTGGTTTCACTGGATGTTGCAGCGCATGAAGTTAGCCATGGTTTTACCGAACAGAATAGCGGTCTGATCTATCGTGGCCAGTCAGGCGGCATGAATGAAGCATTTTCAGATATGGCGGGTGAAGCAGCGGAATTTTATATGCGTGGCAAAAATGATTTCCTGATTGGCTATGATATCAAAAAAGGCTCAGGCGCGCTGCGTTATATGGATCAGCCGTCACGTGATGGCCGTAGCATTGATAATGCATCACAGTATTATAACGGCATTGATGTTCATCATAGCTCAGGTGTTTATAATCGTGCATTTTATCTGCTGGCGAATAGCCCGGGCTGGGATACACGCAAAGCGTTTGAAGTGTTTGTGGATGCGAATCGCTATTATTGGACCGCAACATCAAATTATAACAGCGGCGCATGCGGCGTGATTTCAAGCGCACAGAATCGTAATTATAGCGCGGCAGATGTGACCCGCGCGTTTAGCACAGTGGGCGTGACCTGTCCGTCAGCACTCGAG

[0126] SEQ ID NO.2

[0127] MAEAGGPGGNQKIGKYTYGSDYGPLIVNDRCEMDDGNVITVDMNGSTNDSKTTPFRFACPTNTYKQVNGAYSPLNDAHFFGGVVFNLYRDWFGTSPLTHKLYMKVHYGSRVENAYWDGTAmLFGDGATMFYPLVSLDVAAHEVSHGFTEQN SGLIYRGQSGGMNEAFSDMAGEAAEFYMRGKNDFLIGYDIKKGSGALRYMDQPSRDGRSIDNASQYYNGIDVHHSSGVYNRAFYLLANSPGWDTRKAFEVFVDANRYYWTATSNYNSGACGVISSAQNRNYSAADVTRAFSTVGVTCPSALE

[0128] SEQ ID NO.3

[0129] GCGGAAGCGGGCGGTCCTGGCGGTAATCAGAAAATTGGCAAATATACATACGGTAGCGATTATGGCCCGCTGATTGTGAATGATCGCTGCGAAATGGATGATGGTAATGTGATTACCGTTGATATGAATGGCTCAACCAATGATTCAAAAACAACACCGTTTCGTTTTGCATGCCCGACCAATACATATAAACAGGTGAATGGCGCGTATTCACCGCTGAATGATGCGCATTTCTTTGGTGGCGTTGTTTTTAATCTGTATCGCGATTGGTTTGGTACAAGCCCGCTGACACATAAACTGTATATGAAAGTTCATTACGACCGCTCAGTGGAAAATGCGTATTGGGATGGTACAGCGATGCTGTTCGGGAACGGCGCGACAATGTTTTATCAGCTGGTTTCACTGGATGTTGCAGCGCACGAAGTTAGCCATGGTTTTACCGAACAGAATAGCGGTCTGATCTATCGTGGCCAGTCAGGCGGCATGAATGAAGCATTTTCAGATATGGCGGGTGAAGCAGCGGAATTTTATATGCGTGGCAAAAATGATTTCCTGATTGGCTATGATATCAAAAAAGGCTCAGGCGCGCTGCGTTATATGGATCAGCCGTCACGTGATGGCCGTAGCATTGATAATGCATCACAGTATTATAACGGCATTGATGTTCATCATAGCTCAGGTGTTTATAATCGTGCATTTTATCTGCTGGCGAATAGCCCGGGCTGGGATACACGCAAAGCGTTTGAAGTGTTTGTGGATGCGAATCGCTATTATTGGACCGCAACATCAAATTATAACAGCGGCGCATGCGGCGTGATTTCAAGCGCACAGAATCGTAATTATAGCGCGGCAGATGTGACCCGCGCGTTTAGCACAGTGGGCGTGACCTGTCCGTCAGCA

[0130] SEQ ID NO.4

[0131] AEAGGPGGNQKIGKYTYGSDYGPLIVNDRCEMDDGNVITVDMNGSTNDSKTTPFRFACPTNTYKQVNGAYSPLNDAHFFGGVVFNLYRDWFGTSPLTHKLYMKVHYDRSVENAYWDGTAmLFGNGATMFYQLVSLDVAAHEVSHGFTEQNSGLIYRGQSGGMNEAFSDMAGEAAEFYMRGKNDFLIGYDIKKGSGALRYMDQPSRDGRSIDNASQYYNGIDVHHSSGVYNRAFYLLANSPGWDTRKAFEVFVDANRYYWTATSNYNSGACGVISSAQNRNYSAADVTRAFSTVGVTCPSA

[0132] SEQ ID NO.5

[0133] ATGTGTGTTAAGAAGAAGAACGTTATGACCAGTGTTTTATTGGCAGTTCCATTATTGTTTTCAGCTGGCTTTGGCGGCTCAATGGCTAATGCT

[0134] SEQ ID NO.6

[0135] GGACTA GTGCGGAAGCGGGCGGTC(Spe I)

[0136] SEQ ID NO.7

[0137] TCCCCGCGGTTACTCGAGTGCTGA CGGACA (Sac II)。

Claims

1. A carrier, characterized in that The vectors include vector A, vector B and vector C; wherein vector A is obtained by connecting kerD21 and pHT-01-L, and is denoted as pHT-01-D21, wherein the nucleotide sequence of kerD21 is shown in SEQ ID NO.1; Vector B is based on plasmid pHT-01-D21 connected with a signal peptide; the signal peptide is SP Q7 , whose encoding is SP Q7 The nucleotide sequence of the gene is shown in SEQ ID NO.5; Vector C is obtained by connecting pHT-01-KerD21124D-124N 126A-126E with a signal peptide; the nucleotide sequence of the pHT-01-KerD21124D-124N 126A-126E encoding gene is shown in SEQ ID NO.3, and the signal peptide is SP Q7 , whose encoding is SP Q7 The nucleotide sequence of the gene is shown in SEQ ID NO.

5.

2. A recombinant engineered bacterium carrying the vector according to claim 1.

3. A method for preparing a highly active keratinase, characterized in that: The recombinant engineering bacteria according to claim 2 are inoculated into a fermentation medium for cultivation and then the supernatant is collected by centrifugation to obtain a highly active keratinase.

4. The method for preparing a highly active keratinase according to claim 3, characterized in that: The following steps are involved: (1) The keratinase gene of Pseudomonas aeruginosa PAD21 was selected, and codon optimization was performed using Bacillus subtilis as a host. The codon-optimized recombinant keratinase gene was named kerD21, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2; (2) The pHT-01 vector is double-digested with restriction endonucleases to obtain a linearized vector pHT-01-L; the kerD21 gene described in step (1) is used as a template to perform PCR amplification to obtain kerD21 containing restriction sites SpeI and SacII, and then kerD21 is connected to pHT-01-L and transformed into Escherichia coli (E. coli DH5α) recipient bacteria, and the recombinant shuttle plasmid is obtained by extracting the plasmid and recorded as pHT-01-D21. Finally, pHT-01-D21 is transformed into Bacillus subtilis WB800N to obtain an engineered bacterium that can be used for exocrine expression and production of keratinase KerD21, which is recorded as B. subtilis WB800N-pHT-01-D21; (3) inoculating the recombinant keratinase engineered bacteria B. subtilis WB800N-pHT-01-D21 obtained in step (2) into LB medium, culturing for a period of time, centrifuging the fermentation liquid obtained after the cultivation, and separating the bacteria, and the obtained supernatant is recorded as KerD21; (4) Using the kerD21 gene described in step (1) as a template, error-prone PCR amplification was performed to obtain the target fragment kerD21 eq , and kerD21 eq kerD21 was obtained by double restriction digestion eq-c kerD21 was ligated using T4 DNA ligase eq-c Connect to the linearized fragment of the expression vector pHT-01-L, and then transform the E. coli (E. coli DH5α) recipient bacteria. After transformation, the recombinant shuttle plasmid library is obtained by co-extraction, which is recorded as pHT-01-D21 RM Finally, pHT-01-D21 RM Co-transformed Bacillus subtilis WB800N to obtain a recombinant keratinase mutant strain library that can be used for secretory expression screening, denoted as B. subtilisWB800NpHT-01-D21 RM ; (5) Establishing a high-throughput screening method for keratinase KerD21 mutants: First, prepare a milk screening medium, wherein the milk screening plate is prepared by adding 1-1.5% skim milk powder to LB solid medium, and controlling the content of peptone to be no more than 6%; prepare a milk screening plate based on the milk screening medium, and then add the B. subtilis WB800N-pHT-01-D21 obtained in step (4) RM The transformants were spread on milk screening plates. After culture, the sizes of the transparent zones were compared, and the ratio of the transparent zone to the colony area was calculated. The mutant with the highest ratio was recorded as KerD21 M, and the corresponding recombinant engineered bacteria was recorded as B. subtilisWB800NpHT-01-D21-M. (6) First, the recombinant engineered bacteria B. subtilis WB800N pHT-01-D21-M was inoculated into a 96-well plate containing LB liquid culture medium, and the 96-well plate was sealed with a sealing film to culture the bacteria to obtain a cultured bacterial solution; The cultured bacterial liquid was inoculated into a 96-well deep-well plate containing LB medium for cultivation, and the supernatant was collected by centrifugation after cultivation; The supernatant was added to a 96-well deep-well plate, and feather powder and Tris-HCl buffer were added for mixed reaction. After the reaction, trichloroacetic acid was added to terminate the reaction. The supernatant was collected by centrifugation and the OD of the supernatant was measured. 280nm The mutant with the highest absorption value was recorded as KerD21124D-124N 126A-126E, and the corresponding expression vector was recorded as pHT-01-KerD21124D-124N 126A-126E. The nucleotide sequence of its encoding gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; (7) Based on the pHT-01-D21 obtained in step (2), the signal peptide was connected to pHT-01-D21 to construct a pHT-01-D21-SP vector, and the Escherichia coli (E. coli DH5α) recipient bacteria were transformed; the recombinant signal peptide shuttle plasmid was obtained by extraction, and then the recombinant signal peptide shuttle plasmid was transformed into Bacillus subtilis WB800N to obtain a recombinant keratinase signal peptide library that can be used for secretory expression screening, which was recorded as B. subtilis WB800NpHT-01-D21-SP; (8) The same procedure as in step (3) was followed, except that B. subtilis WB800N pHT-01-D21-SP was replaced with B. subtilis WB800N-pHT-01-D21; the resulting supernatant was designated KerD21-SPS; KerD21-SPS was mixed with Tris-HCl buffer and feather powder for reaction. After the reaction, trichloroacetic acid was added to terminate the reaction and the OD was measured. 280nm The mutant with the highest absorbance value was selected as the best mutant, and its corresponding signal peptide was the best signal peptide. The best signal peptide was SP Q7 ; Its code SP Q7 The nucleotide sequence of the gene is shown in SEQ ID NO.5; (9) SP was ligated by double enzyme digestion method Q7 Connect with the expression vector pHT-01-KerD21124D-124N126A-126E obtained in step (6) to obtain a highly efficient signal peptide SP Q7 The KerD21124D-124N 126A-126E mutant expression vector was labeled pHT-01-KerD21124D-124N 126A-126E (SP Q7 ); and pHT-01-KerD21124D-124N126A-126E (SP Q7 ) was transformed into Bacillus subtilis WB800N to obtain a recombinant engineered bacterium, which was recorded as B. subtilis WB800N-pHT-01-KerD21124D-124N 126A-126E (SP Q7 ); Finally, follow the steps in step (3), except that B. subtilis WB800N-pHT-01-KerD21124D-124N126A-126E (SP Q7 ) was replaced with B. subtilis WB800N-pHT-01-D21; the resulting supernatant was a highly active keratinase, denoted as KerD21124D-124N 126A-126E (SP Q7 ).

5. The method for preparing a highly active keratinase according to claim 4, characterized in that: In step (3), the inoculation amount of the recombinant keratinase engineering bacteria inoculated into the LB culture medium is 1%; the culture conditions are 37° C., 200 rpm, and culture for 12-14 hours; The culture conditions in step 5 are: 37° C., 200 rpm, 10-12 h; the milk screening plate; contains components by mass fraction: 1.5% skim milk powder, 5% peptone, 5% yeast extract powder, 10% NaCl, and 10-15% agar; The conditions for bacterial culture in step 6 are: placing in a constant temperature shaking shaker at 37° C. and 180 rpm for overnight culture; the inoculation amount of the cultured bacterial solution inoculated into the LB medium is 1%; the culture conditions are 37° C. and 200 rpm for 10-14 h; the centrifugation conditions are: centrifugation at 4000 rpm for 15 min; the reaction time is 1 h, and the temperature is 37-40° C.; the amount relationship of the feather powder, supernatant, Tris-HCl buffer and trichloroacetic acid is 0.0094 g: 0.2 mL: 0.4 mL: 0.4 mL; wherein the pH of the Tris-HCl buffer is 7.0, the concentration is 0.05 mol / L, and the concentration of trichloroacetic acid is 10%; The reaction time in step 8 is 1 hour, and the temperature is 37-40° C.; the dosage of KerD21-SPS, Tris-HCl buffer, feather powder and trichloroacetic acid is 1 mL: 2 mL: 0.047 g: 2 mL; the pH of Tris-HCl buffer is 7.0, the concentration is 0.05 mol / L, and the concentration of trichloroacetic acid is 10%.

6. A highly active keratinase prepared according to the method of claim 3 or 4.

7. Use of the highly active keratinase for feather powder degradation according to claim 6, characterized in that: The steps are as follows: Use feather powder as raw material, add keratinase crude enzyme solution, and cooperate with Mg 2+ forming a mixed system; controlling the pH of the mixed system, and reacting under certain temperature conditions to degrade keratin in feathers; the crude keratinase solution includes KerD21, KerD21124D-124N 126A-126E and KerD21124D-124N 126A-126E (SP Q7 ).

8. The use according to claim 7, characterized in that Based on the volume fraction of the mixed system, the amount of crude keratinase added is 1%-25% of the mixed system. 2+ The concentration of Mg is 1-5mM; the reaction temperature is 37℃ and the reaction time is 1h; 2+ Including MgCl2.

9. The use of the highly active keratinase according to claim 6 for preparing feather meal fermented feed, characterized in that: The method comprises the following steps: firstly, mixing feather powder, bean dregs and second-rate powder to obtain a mixed raw material; then inoculating fermented seed liquid of saccharomyces cerevisiae, bacillus licheniformis, lactobacillus fermentation and lactobacillus rhamnosus, adding crude keratinase enzyme liquid, methionine, lysine and mineral ions, and finally adding water to a target water content, performing anaerobic fermentation and obtaining fermented feed after the fermentation is completed; the crude keratinase enzyme liquid comprises KerD21, KerD21124D-124N 126A-126E and KerD21124D-124N126A-126E(SP Q7 ).

10. The use according to claim 9, characterized in that The mass ratio of feather meal, bean dregs and second-grade powder is 5:3:2, the total inoculation amount of fermentation bacterial liquid of saccharomyces cerevisiae, bacillus licheniformis, lactobacillus fermentation and lactobacillus rhamnosus is 6% of the mass of the mixed raw materials, and the volume ratio of fermentation seed liquid of saccharomyces cerevisiae, bacillus licheniformis, lactobacillus fermentation and lactobacillus rhamnosus is 2:2:1:1; The mineral ions include CaCO3, which is added in an amount of 2% of the mass of the mixed raw materials; methionine is added in an amount of 0.20-0.30% of the total mass of the mixed raw materials, and lysine is added in an amount of 0.25% of the mass of the mixed raw materials; The addition amount of the crude keratinase solution is 1%-15% of the mass of the mixed raw materials; the target water content is 35-45%; the fermentation temperature is 35-40°C, and the fermentation time is 7 days.

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