Preparation method of acid-resistant high-activity keratinase preparation and application of acid-resistant high-activity keratinase preparation in feather meal fermented feed

Abstract

The invention belongs to the field of enzyme engineering and agricultural waste conversion, and particularly relates to a preparation method of an acid-resistant high-activity keratinase preparation and application of the acid-resistant high-activity keratinase preparation in feather meal fermented feed. According to the invention, acid resistance design is carried out based on cotG-KERQ7, and the acid stability and activity of the keratinase mutant are both improved through a combined mutation strategy of three rounds of base catalytic residue replacement in a catalytic center, surface unstable positive charge amino acid replacement and flexible region introduction. By making the zymoprotein surface of serine keratinase more suitable for an acidic environment, the volume of a substrate binding cavity of protease in the acidic environment is increased, and the substrate can be better accepted and catalyzed, so that the acid stability and activity are effectively improved; finally, the acid-resistant and high-activity keratinase preparation B.subtilis cotG-KERQ7-M2 is obtained, and reference is provided for subsequent industrialized and fermented feed preparation for green conversion of feather waste into acid-soluble protein.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering and agricultural waste conversion, and particularly relates to a preparation method of an acid-resistant high-activity keratinase preparation and application of the preparation in feather meal fermented feed. Background Art

[0002] According to statistics, the global poultry industry produces up to 80 million tons of feather waste each year. If these feather wastes are landfilled, it will not only lead to a huge waste of protein resources, but also cause environmental pollution and the spread of harmful bacteria in poultry. The main component of feathers is β-keratin, which is rich in amino acids such as cystine, lysine, proline and serine, as well as a series of trace elements. Compared with α-keratin, β-keratin contains more cysteine ​​residues and disulfide bonds, thus having higher mechanical strength. A large number of hydrophobic amino acids are distributed on the periphery of keratin, while hydrophilic amino acids and groups are mainly embedded in the protein. Due to the highly cross-linked disulfide bonds, hydrogen bonds and hydrophobic interactions between molecules, it is difficult for traditional proteases (such as trypsin, pepsin and papain) to effectively degrade it. This leads to a decrease in protein quality and a decrease in the digestibility of feather meal, so feather meal is considered to be a low-quality feed. The traditional feed industry produces feather meal by high temperature and high pressure treatment of discarded feathers. Not only do some amino acids deteriorate, but the different chemical groups of feather meal keratin are not significantly disassembled in structure, making it difficult to be digested by conventional proteases.

[0003] Keratinase acts on keratin with a highly cross-linked structure and can also directly act on proteins rich in hydrophobic amino acids. Therefore, keratinase can be added to feather meal feed to obtain a large amount of feed peptides and amino acids. It is reported that Adejumo et al. isolated a strain of Bifidobacterium licheniformis LMUB05 from a feather dumping site, which can produce keratinase. They used it to treat feathers to obtain hydrolyzates and added them to broiler feed to observe the effects on poultry production performance. It was found that there was no significant difference between the broiler feeding effect and the standard feed. In general, the addition of keratinase can improve the digestibility and palatability of feather meal and other hydrophobic feed protein raw materials in animals, and have better intestinal flora. The currently popular lactic acid bacteria fermented feed can not only enhance the nutritional value and absorption rate of the feed itself, but also effectively improve the palatability of the feed. The production of a large amount of organic acids during the fermentation process will cause the feed environment to be acidic, and the pH of lactic acid bacteria fermented feed is a key indicator for evaluating the fermentation quality. The pH of well-fermented feed should be 4.2 or lower. However, most keratinase prefers a neutral to alkaline environment with a pH between 7 and 10, and there are very few studies on acid-resistant keratinase in the world. In order for keratinase to remain stable and efficient in enzymolysis in lactic acid bacteria fermented feed and in the animal stomach, keratinase needs to have good acid resistance.

[0004] In recent years, the modification of keratinase has become a research hotspot. The thermal stability of keratinase can be improved by designing highly flexible loop regions, enhancing the binding to Ca 2+ The binding capacity of keratinase is achieved by adjusting the binding capacity of keratinase, etc. However, it has always been a challenge to shift the optimal pH of keratinase toward acidity or to improve the activity of keratinase under acidic conditions. According to the catalytic type classification, the keratinase KERQ7 explored in this study belongs to the serine protease family. This serine protease uses a catalytic mechanism called a catalytic triad. Its typical catalytic triad is a Ser-His-Asp hydrogen bond network, in which the Ser residue attacks the carbonyl carbon as a nucleophile, and His extracts a proton from the catalytic Ser to enhance the nucleophilicity of the seryl residue. Serine proteases with a typical catalytic triad usually show the highest activity at neutral to slightly alkaline pH values. The catalytic activity of most enzymes is pH-dependent, and the hydrogen bonds in the center of the enzyme active site are usually very sensitive to the environmental pH value, which also makes the design of the optimal pH of the enzyme a very challenging task. Therefore, it is necessary to establish a general and effective protein design strategy to redesign the pH-related amino acid residues of the enzyme to regulate the acid resistance of serine family keratinase.

[0005] Microbial surface display is a technology that uses genetic engineering to locate foreign proteins or peptides on the surface of bacteriophages, yeast or spores. Bacillus subtilis is an internationally recognized food-grade expression strain (GRAS). Its spores, as a biological carrier, provide an ideal platform for enzyme fixation due to its safety, stability and easy preparation. Immobilizing enzymes on spores not only enables the enzymes to resist various harsh environments, thereby meeting the stringent requirements of industrial applications, but also greatly improves the stability and durability of the enzymes. Spore surface display uses the anchoring effect of capsid proteins to directly fix exogenous proteins on the surface of spores, avoiding the barrier of the cytoplasmic membrane. In addition, the enzymes displayed by spores are easier to purify and concentrate, and purer spore enzyme preparations can be prepared by simple centrifugation or filtration, which reduces the purification cost and can be widely used in industrial and agricultural outputs. Summary of the invention

[0006] In order to solve the problem of waste of rich protein resources in discarded feathers and the difficulty of conventional keratinase to maintain high activity in the acidic environment of the fermented feed industry, the present invention uses a keratinase KERQ7 anchored on the capsid protein cotG on the surface of Bacillus subtilis DB403 as the starting target for acid resistance modification. By changing the acid-base catalytic residues in the catalytic triad, mutating the unstable positively charged amino acids on the surface, introducing a flexible ring, and through computational design and experimental verification, a keratinase mutant that still has high activity in an acidic environment was successfully constructed, and it was applied to lactic acid bacteria fermented feather powder feed, and the fermentation parameters were measured to identify the acid resistance improvement of keratinase molecular evolution mutants. Efficient enzymatic hydrolysis of feather powder can significantly reduce the cost of feed protein, thereby promoting the green and efficient development of the feed industry and aquaculture industry.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0008] The present invention first provides a KERQ7 keratinase mutant fragment, wherein the KERQ7 keratinase mutant fragment is Q7-M2, the gene nucleotide sequence of Q7-M2 is shown in SEQ ID NO.7, and the amino acid sequence encoded by it is shown in SEQ ID NO.8. Accordingly, a plasmid vector carrying the KERQ7 keratinase mutant fragment is provided; and further a recombinant engineered bacterium carrying the gene or the plasmid vector is provided.

[0009] The present invention also provides a method for preparing an acid-resistant high-activity keratinase preparation, which comprises the following steps: inoculating the recombinant engineered bacteria into a fermentation medium, culturing the recombinant bacteria, collecting the bacterial bodies by centrifugation, and resuspending the bacteria in a PBS buffer to obtain a recombinant spore protein suspension with keratinase displayed on the surface; or collecting the bacterial bodies and subjecting them to low-temperature drying to obtain a solid powder; the recombinant spore protein suspension or the solid powder are both acid-resistant high-activity keratinase preparations, which are recorded as B. subtilis cotG-KERQ7-M2.

[0010] The preparation method of the acid-resistant high-activity keratinase preparation comprises the following specific steps:

[0011] (1) The present invention first selects keratinase KERQ7 (sequence number: KP694221) displayed on the surface of anchor protein cotG, denoted as cotG-KERQ7, wherein the nucleotide sequence of the mature fragment of KERQ7 is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2;

[0012] Furthermore, the steps for obtaining cotG-KERQ7 in step (1) are as follows: using the sequence SEQ ID NO.1 of KERQ7 as a template, amplifying KERQ7 with primers Q7-F and Q7-R to obtain an amplified KERQ7 fragment, and then inserting the KERQ7 fragment into the cotG plasmid using two endonucleases, Spe I and HindIII, to construct a recombinant plasmid, recorded as cotG-KERQ7; wherein the amplification conditions are: 94°C for 3 min, 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and 30 cycles.

[0013] (2) integrating the cotG-KERQ7 described in step (1) into the Bacillus subtilis shuttle expression vector pHS to form a shuttle plasmid pHS-cotG-KERQ7;

[0014] (3) Using the shuttle plasmid pHS-cotG-KERQ7 described in step (2) as a template, performing one-step whole plasmid PCR site-directed mutagenesis to change the catalytic residue His64 to Glu64, thereby obtaining a mutant plasmid pHS-cotG-KERQ7-64E, which is recorded as pHS-cotG-KERQ7-64E, wherein the KERQ7-64E keratinase mutant fragment in the mutant plasmid pHS-cotG-KERQ7-64E is recorded as KERQ7-64E; the nucleotide sequence of KERQ7-64E is shown in SEQ ID NO.3, and the amino acid sequence encoded by it is shown in SEQ ID NO.4; finally, pHS-cotG-KERQ7-64E is transformed into Escherichia coli DH5α, and the DH5α-pHS-cotG-KERQ7-64E complete plasmid is extracted;

[0015] Furthermore, the step of obtaining pHS-cotG-KERQ7-64E in step (3) is as follows: using the shuttle plasmid pHS-cotG-KERQ7 as a template, using primers 64E-F and 64E-R to perform one-step PCR amplification of the pHS-cotG-KERQ7 plasmid, and finally obtaining a mutant plasmid, which is recorded as pHS-cotG-KERQ7-64E;

[0016] The PCR amplification conditions were: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, and 30 cycles; the sequence of 64E-F was 5'-ccgaatgcgacacaagattttcaatca gag gcacaacatgttgc-3'; the sequence of 64E-R is 5'-gcaacatgttgtgc ctc tgattgaaaatcttgtgtcgcattcgg-3'.

[0017] (4) Using the amino acid sequence of the keratinase mutant fragment KERQ7-64E in step (3) as a template, a three-dimensional protein model is constructed, and the obtained protein model is recorded as Q7-64E; Q7-64E is then molecularly docked with the keratin analog AAPF (N-succinyl-ala-ala-pro-phe-p-nitroanilide, PubChem CID 5496888) to obtain a Q7-64E-AAPF protein ligand complex, and a molecular dynamics simulation is performed on the complex in an acidic pH environment. The unstable, positively charged amino acid sites located on the surface of the enzyme protein during the catalytic process can be obtained through the molecular dynamics simulation results; after simulating the Q7-64E-AAPF protein ligand complex in an acidic pH environment, the root mean square deviation (RMSD) of the protein three-dimensional structure model under different pH environments is The change trend of the root mean square deviation is different. The root mean square fluctuation value of amino acids at each site of the protein three-dimensional structure is different under different pH environments. The evaluation of its stability is based on the height of the trend line of the root mean square deviation. The lower the more stable. Therefore, the protein three-dimensional structure model with the lowest root mean square deviation is selected and recorded as

[0018] Based on the root mean square fluctuation value of each amino acid site of model 64E as a reference, the modification sites were selected; the modification sites included surface amino acids Arg145, Arg98, His36 and His17;

[0019] Preferably, Alphafold2 is selected for construction when constructing the protein three-dimensional model in step (4); the molecular docking of Q7-64E and the keratin analog AAPF is performed by Autodock, and the docking posture selects the conformation in which the catalytic amino acid Ser221 of Q7-64E directly interacts with the AAPF amide bond; wherein the acidic pH environment in the molecular dynamics simulation is 4-6, and the pH environment corresponding to the lowest root mean square deviation is 5.

[0020] (5) Using DH5α-pHS-cotG-KERQ7-64E obtained in step (3) as a template, performing one-step whole plasmid PCR site-directed mutagenesis to change the surface amino acid Arg145 to Glu145, thereby obtaining a mutant plasmid pHS-cotG-KERQ7-145E, recorded as pHS-cotG-KERQ7-145E, and then transforming pHS-cotG-KERQ7-145E into Escherichia coli DH5α, and extracting to obtain the complete plasmid DH5α-pHS-cotG-KERQ7-145E;

[0021] Furthermore, the steps for obtaining pHS-cotG-KERQ7-145E in step (5) are as follows: using plasmid DH5α-pHS-cotG-KERQ7-64E as a template, using primers 145E-F and 145E-R to amplify pHS-cotG-KERQ7-64E plasmid by one-step PCR, and finally obtaining a mutant plasmid, which is recorded as pHS-cotG-KERQ7-145E;

[0022] The PCR amplification conditions were as follows: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min for 30 cycles;

[0023] The sequence of 145E-F is 5'-ttaaaaatgcggtggatacagcgaataat gag ggcgttgttgttgttgttgcag-3'; the primer sequence for 145E-R is 5'-ctgcaacaacaacaacgcc ctc attattcgctgtatccaccgcatt-3'.

[0024] (6) Preparation of mutant plasmid pHS-cotG-KERQ7-M1;

[0025] S1. Using DH5α-pHS-cotG-KERQ7-145E obtained in step (5) as a template, performing one-step full-plasmid PCR site-directed mutagenesis to change the surface amino acid Arg98 to Glu98, thereby obtaining a mutant plasmid pHS-cotG-KERQ7-98E, recorded as pHS-cotG-KERQ7-98E, transforming pHS-cotG-KERQ7-98E into Escherichia coli DH5α, and extracting to obtain the complete plasmid DH5α-pHS-cotG-KERQ7-98E;

[0026] Furthermore, the step of obtaining pHS-cotG-KERQ7-98E in S1 of step (6) is as follows: using plasmid DH5α-pHS-cotG-KERQ7-145E as a template, using primers 98E-F and 98E-R to amplify pHS-cotG-KERQ7-145E plasmid by one-step PCR, and finally obtaining a mutant plasmid, which is recorded as pHS-cotG-KERQ7-98E;

[0027] The conditions for PCR amplification were as follows: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min, for 30 cycles;

[0028] The primer sequence of 98E-F is: 5'-gcctgtatgcggtgaaagttcttgat gagaatggagatggccaat-3'; the primer sequence of 98E-R is: 5'-ccatgaatattggccatctccatt ctc atcaagaactttcaccgcatacag-3'.

[0029] S2. Using DH5α-pHS-cotG-KERQ7-98E obtained in S1 as a template, perform one-step whole-plasmid PCR site-directed mutagenesis to change the surface amino acid His36 to Glu36 to obtain the mutant plasmid pHS-cotG-KERQ7-36E, recorded as pHS-cotG-KERQ7-36E, transform pHS-cotG-KERQ7-36E into Escherichia coli DH5α, and extract to obtain the complete plasmid DH5α-pHS-cotG-KERQ7-36E.

[0030] Furthermore, the step of obtaining pHS-cotG-KERQ7-36E in S2 of step (6) is as follows: using plasmid DH5α-pHS-cotG-KERQ7-98E as a template, using primers 36E-F and 36E-R to amplify pHS-cotG-KERQ7-98E plasmid by one-step PCR, and finally obtaining a mutant plasmid, which is recorded as pHS-cotG-KERQ7-36E;

[0031] The conditions for PCR amplification were: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min for 30 cycles;

[0032] The sequence of 36E-F is: 5'-gcatt gag gcagcacatccggatctgaatgttgcagga-3'; the sequence of 36E-R is: 5'-ggatgtgctgc ctc aatgcctgtatccagaactgccac-3'.

[0033] S3. Using DH5α-pHS-cotG-KERQ7-36E obtained in S2 as a template, a one-step whole plasmid PCR site-directed mutagenesis was performed to change the surface amino acid His17 to Glu17, thereby obtaining a mutant plasmid pHS-cotG-KERQ7-17E, which was recorded as pHS-cotG-KERQ7-M1. The pHS-cotG-KERQ7-M1 was transformed into Escherichia coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-M1 was extracted, wherein the KERQ7 keratinase mutant fragment was recorded as Q7-M1, the nucleotide sequence of Q7-M1 was shown in SEQ ID NO.5, and the amino acid sequence encoded by it was shown in SEQ ID NO.6;

[0034] Furthermore, the step of obtaining pHS-cotG-KERQ7-M1 in S3 of step (6) is as follows: using plasmid DH5α-pHS-cotG-KERQ7-36E as a template, using primers 17E-F and 17E-R to amplify pHS-cotG-KERQ7-36E plasmid by one-step PCR, and finally obtaining a mutant plasmid, which is recorded as pHS-cotG-KERQ7-M1;

[0035] The conditions for PCR amplification were: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min for 30 cycles;

[0036] The sequence of 17E-F is 5'-ggcagtg gag gcgcaaggatataaaggagcaaatgt-3'; the sequence of 17E-R is 5'-ccttgcgc ctc cactgccggatccttaatctgttcctg-3'.

[0037] S4. Using the amino acid sequence of the keratinase mutant fragment Q7-M1 in S3 as a template, a three-dimensional protein model was constructed, and the resulting protein model was recorded as KERQ7-M1; KERQ7-M1 was then molecularly docked with the keratin analog AAPF (N-succinyl-ala-ala-pro-phe-p-nitroanilide, PubChem CID 5496888) to obtain the KERQ7-M1-AAPF protein ligand complex, and the complex was subjected to molecular dynamics simulation in an acidic pH environment; after simulating the KERQ7-M1-AAPF protein ligand complex in an acidic pH environment, the three-dimensional structure model of the protein was affected by different environmental pH under different pH environments, and the root mean square fluctuation of amino acids at each site changed differently. The change in RMSF was used as the standard for evaluating the effect of the acidic environment on the complex.

[0038] Combined with the RMSF results of molecular dynamics simulation of KERQ7-M1-AAPF in pH environment, the region outside the catalytic pocket of KERQ7-M1 that is most affected by the change in acidic environment is selected, that is, the region with the largest change in RMSF value, which is Glu36~Asn43;

[0039] Preferably, Alphafold2 is selected for construction when constructing the three-dimensional protein model in S4; KERQ7-M1 and the keratin analog AAPF are molecularly docked by Autodock, and the docking posture selects the conformation in which the catalytic amino acid Ser221 of KERQ7-M1 directly interacts with the amide bond of AAPF; the acidic pH environment in the molecular dynamics simulation is 4-6.

[0040] (7) Based on the region Glu36 to Asn43 selected in step (6), Glu36 is then used as the insertion site to insert the AAEPVLDTGIE amino acid fragment after the Glu36 site of the keratinase mutant KERQ7-M1 on the pHS-cotG-KERQ7-M1 plasmid to obtain a complete mutant plasmid with the new insertion fragment, which is recorded as pHS-cotG-KERQ7-M2; pHS-cotG-KERQ7-M2 is then transformed into Escherichia coli DH5α, and the DH5α-pHS-cotG-KERQ7-M2 complete plasmid is extracted, wherein the KERQ7 keratinase mutant fragment is recorded as Q7-M2, the nucleotide sequence of Q7-M2 is shown in SEQ ID NO.7, and the amino acid sequence encoded by it is shown in SEQ ID NO.8;

[0041] (8) The complete plasmid DH5α-pHS-cotG-KERQ7-M2 obtained in step (7) is transformed into Bacillus subtilis DB403 to obtain an engineered bacterium B. subtilis DB403 pHS-cotG-KERQ7-M2 that can be used to display keratinase on the surface of spores; the engineered bacterium B. subtilis DB403 pHS-cotG-KERQ7-M2 is inoculated into LB medium to obtain a culture solution after a first culture; the culture solution is then inoculated into DSM liquid medium, and after a second culture, the bacterial precipitate is collected by centrifugation, and the precipitate is resuspended in GTE Buffer containing lysozyme to obtain a resuspended solution, and the precipitate is collected by centrifugation after incubation and washed with PBS Buffer. After washing, the precipitate is collected and resuspended in PBS buffer (PBS Buffer) again to obtain a recombinant spore protein suspension with keratinase displayed on the surface; or the precipitate collected after washing is subjected to low-temperature drying to obtain a solid powder; the recombinant spore protein suspension or solid powder is a keratinase preparation, which is recorded as B. subtilis cotG-KERQ7-M2.

[0042] Preferably, the concentration of lysozyme in the GTE Buffer in step (8) is 2 mg / mL, wherein the components of the GTE Buffer are: 10 mM EDTA, 20 mM Tris-HCl, pH = 7.5, 50 mM Glucose;

[0043] The conditions for the first culture were 37°C, 200 rpm, overnight culture (10-12 h); the conditions for the second culture were 37°C, 200 rpm, 36 h; the conditions for the incubation were 37°C, 60 min, the pH of PBS Buffer was 7.4, and the components of PBS Buffer were (based on 1 L): 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4; the OD of the recombinant spore protein suspension was 600nm is 1-2.

[0044] The acid-resistant high-activity keratinase preparation prepared by the present invention is used for preparing feather meal fermented feed, and the steps are as follows:

[0045] The feed raw materials are composed of feather meal, bean dregs and second-rate flour. Firstly, fermented seed liquid of brewer's yeast, bacillus licheniformis, lactobacillus fermentation and lactobacillus rhamnosus is inoculated into the feed raw materials, and then an acid-resistant high-activity keratinase preparation B. subtilis cotG-KERQ7-M2, methionine (Met) and lysine (Lys) are added. Finally, water is added to the target water content to obtain a fermentation system, and synergistic anaerobic fermentation is carried out. After the fermentation is completed, high-quality feather meal fermented feed can be obtained.

[0046] Preferably, the mass ratio of feather meal, bean dregs and inferior powder is 4:3:3; the total inoculation amount of fermented seed liquid of Saccharomyces cerevisiae, Bacillus licheniformis, Lactobacillus fermentation and Lactobacillus rhamnosus is 6% (mL / g; that is, the total inoculation amount of fermented seed liquid and the dosage relationship of feed raw materials are 6mL:100g), wherein the volume ratio of fermented seed liquid of Saccharomyces cerevisiae, Bacillus licheniformis, Lactobacillus fermentation and Lactobacillus rhamnosus is 2:2:1:1; the addition amount of acid-resistant high-activity keratinase preparation B.subtilis cotG-KERQ7-M2 is as follows: when using spore suspension, the addition amount is 5-15% of the mass of feed raw materials (that is, the dosage relationship of spore suspension and feed raw materials is 10-15mL:100g); when using solid powder, the addition amount of powder is 0.5-1.5% of the mass of feed raw materials.

[0047] The added amount of methionine is 0.20-0.30% of the mass of the feed raw material, and the added amount of lysine is 0.25% of the mass of the feed raw material; the target water content is 35-45%; the fermentation temperature is 25-40° C., and the fermentation time is 2-14 days.

[0048] The fermentation method of the feed is: the obtained fermentation system is mixed evenly and then placed in a one-way valve anaerobic fermentation bag for one-step constant temperature fermentation.

[0049] The advantages and technical effects of the present invention are:

[0050] (1) The present invention establishes a rational design idea for the acid resistance of keratinase displayed by a three-step spore method. As one of the very few studies on acid resistance modification of keratinase, the present invention is original. In this study, the acid resistance mutation of keratinase cotG-KERQ7 displayed on the spore surface was firstly laid by replacing the alkaline catalytic residues in the active center. Then, the molecular dynamics simulation of the enzyme-substrate complex KERQ7-AAPF was carried out to enhance the acid stability and activity by replacing unstable positively charged amino acids on the surface. Finally, based on the rigidity-flexibility changes of the protein surface in different pH environments, a flexible tentacle was introduced outside the catalytic pocket to enhance the feather powder degradation activity of KERQ7 in an acidic environment; finally, M2 was obtained through the three-step design of the keratinase surface. The spore-displayed mutant M2 can better decompose feather powder in the lactic acid bacteria fermentation feed environment, providing a technical solution for promoting the property improvement and industrial application of keratinase.

[0051] (2) The present invention utilizes the above three-step method for rationally designing the acid resistance of keratinase on the surface of spores to achieve directional modification of the acid resistance of keratinase, and finally obtains a highly efficient spore enzyme preparation with significantly improved activity and acid resistance. In addition, the spore enzyme preparation prepared by the present invention can be directly added and used in the form of a spore suspension, or can be made into a solid powder by low-temperature drying, making it easier to store and transport.

[0052] (3) The present invention discloses a fermentation formula and fermentation process of a feather meal lactic acid bacteria fermented feed with balanced nutrition and good palatability, and uses the prepared keratinase B. subtilis cotG-KERQ7-M2 spore enzyme preparation for bacterial enzyme synergistic fermentation, which significantly increases the content of small peptides and free amino acids in the feather meal feed, and promotes the application of waste feather green conversion protein resources in the feed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Bioinformatics and enzyme activity analysis of mutant 64E; (a) interactions near the catalytic domain, blue dashed lines represent hydrogen bonds, and gray dashed lines represent van der Waals forces; (b) schematic diagram of hydrogen bonds between the catalytic center and the substrate AAPF; (c) RMSD diagram of molecular dynamics simulation of 64E-AAPF complex simulated at different pH; (d) RMSF diagram of molecular dynamics simulation of 64E-AAPF complex simulated at different pH; (e) enzyme activity of wild-type KERQ7 and mutant 64E spore preparations.

[0054] Figure 2The results of the mutation in Example 2 are analyzed; (a) enzyme activity of different spore preparations; (b) comparison of RMSF plots of molecular dynamics simulations of 64E and M1 complexes simulated at different pH values; (c) properties of the substrate binding pockets of 64E and M1.

[0055] Figure 3 is a graph showing the RMSF changes of M1 spore preparations between pH = 4 and 6.

[0056] Figure 4 Comparison of enzyme activities of wild-type KERQ7, M1, and M2 spore preparations.

[0057] Figure 5 It is the residual activity of the enzyme preparation incubated for different time periods under different pH environments; Figure (1) represents the wild-type KERQ7 spore enzyme preparation; Figure (2) represents the KERQ7 mutant M2 spore enzyme preparation.

[0058] Figure 6 Figure 3 is the bioinformatics analysis of mutant M2; (a) the dynamic distance change between the Ser hydroxyl oxygen skeleton and the carbonyl oxygen of the substrate AAPF; (b) the hydrogen bond network between AAPF and M2; (c) the properties of 64E and the oxygen anion hole of M2 (the green part in the red circle is the oxygen anion hole); (d) the RMSF diagram of some residues of M2 and M1; (e) the comparison of the RMSD of the main chains of M1 and M2.

[0059] Figure 7 The effect of spore enzyme preparation on the content of small peptides produced by feather meal fermented feed.

[0060] Figure 8 This is the standard peak diagram of 17 amino acids detected by HPLC; among them: ①Asp②Glu③Ser④Arg⑤Gly⑥Thr⑦Pro⑧Ala⑨Val⑩Met Cys Ile Leu Phe His Lys Tys.

[0061] Figure 9 The effect of spore enzyme preparation on the free amino acid content of feather meal fermented feed. DETAILED DESCRIPTION

[0062] 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, without departing from the spirit and scope of the present invention, can make various modifications to the present invention to adapt it to various uses and conditions.

[0063] E.coli-B.subtilis shuttle expression vector pHS and E.coli DH5α, E.coli BL21, Bacillus subtilis DB403, cotG-KERQ7 are all known. The methods for obtaining the same can refer to patent CN 116926101A or literature on keratinase expression, identification and modification and its application in feather degradation and transformation.

[0064] DSM liquid culture medium (Difco Sporulation Medium), whose components are: 0.8% nutrient broth Difco, 0.1% KCl, 0.025% MgSO4·7H2O, 1.0mM Ca(NO3)2·4H2O, 10μM / LMnCl2, 1.0μM / LFeSO4.

[0065] 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; Bacillus was inoculated into LB medium at 1% inoculum and cultured at 37°C with a shaker 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; obtain the corresponding fermentation seed liquid.

[0066] 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.

[0067] 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 spore enzyme preparation (spore suspension, OD 600nm=1.0), add 2.0 mL of the corresponding pH buffer (sodium hydrogen citrate-citric acid buffer (phosphate buffer) when pH 3-7); then add 10 mg of feather powder as substrate, react in a constant temperature shaker at 30°C or 40°C, add 2.0 mL of 10% trichloroacetic acid (TCA) to terminate the reaction after 1 h, centrifuge at 10000 rpm at 4°C for 10 min, and measure the supernatant at OD 280nm The absorbance at 280 nm was measured, and the reaction tube treated with TCA before the reaction was used as a control. Enzyme activity was defined as an increase of 0.01 in absorbance at 280 nm under the above reaction conditions, which was defined as 1 enzyme activity unit (U).

[0068] Optimization and implementation instructions for related solutions:

[0069] (1) In the implementation process of the present invention, an optimization scheme for displaying KERQ7-M2 enzyme on the surface of spores is involved, and the specific steps are as follows:

[0070] A: Using the pHS-cotG-KERQ7 plasmid as a PCR template, primers 64E-F and 64E-R were used to amplify pHS-cotG-KERQ7 by one-step PCR to obtain the mutant plasmid pHS-cotG-KERQ7-64E. pHS-cotG-KERQ7-64E was digested with DpnI enzyme and heat-shocked transformed into Escherichia coli DH5α competent cells. The DH5α-pHS-cotG-KERQ7-64E plasmid was extracted using a plasmid extraction kit (Omega).

[0071] Primers 145E-F and 145E-R were used to amplify pHS-cotG-KERQ7-64E in one step, and the mutant plasmid DH5α-pHS-cotG-KERQ7-145E was obtained using the same steps as DH5α-pHS-cotG-KERQ7-64E.

[0072] DH5α-pHS-cotG-KERQ7-145E was amplified using 98E-F and 98E-R in one step, and the mutant plasmid DH5α-pHS-cotG-KERQ7-98E was obtained by the same steps.

[0073] DH5α-pHS-cotG-KERQ7-98E was amplified by one-step method using 36E-F and 36E-R, and the mutant plasmid DH5α-pHS-cotG-KERQ7-36E was obtained by the same steps.

[0074] DH5α-pHS-cotG-KERQ7-36E was amplified by one-step method using 17E-F and 17E-R. The mutant plasmid DH5α-pHS-cotG-KERQ7-17E was obtained by the same steps. The mutant plasmid DH5α-pHS-cotG-KERQ7-17E was pHS-cotG-KERQ7-M1 obtained in step (5). The mutation sites introduced into the sequence by each primer are the italic parts in Table 1. The PCR conditions were: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, and 30 cycles.

[0075] B: The recombinant plasmids DH5α-pHS-cotG-KERQ7-64E, DH5α-pHS-cotG-KERQ7-145E, DH5α-pHS-cotG-KERQ7-98E, DH5α-pHS-cotG-KERQ7-36E and DH5α-pHS-cotG-KERQ7-M1 obtained in step A were verified by colony PCR using P1-F and P1-R respectively (PCR conditions were the same as KERQ7 amplification conditions), and then transformed into B. subtilis DB403. The transformants were picked again for colony PCR and fragment sequencing verification, and the verified recombinant Bacillus subtilis were named B. subtilis DB403 pHS-cotG-KERQ7-64E, B. subtilis DB403 pHS-cotG-KERQ7-145E, B. subtilis DB403 pHS-cotG-KERQ7-98E, B. subtilis DB403 pHS-cotG-KERQ7-36E and B. subtilis DB403 pHS-cotG-KERQ7-M1, respectively. pHS-cotG-KERQ7-98E, B. subtilis DB403 pHS-cotG-KERQ7-36E and B. subtilis DB403 pHS-cotG-KERQ7-M1 were cultured and stored at -80°C.

[0076] C: The DH5α-pHS-cotG-KERQ7-M1 verified by PCR and sequenced correctly in step B was submitted to Shanghai Biotech for fragment synthesis and insertion according to the partial sequence (SEQ ID NO.7) of Q7-M2 repetitive expression. After receiving the successfully synthesized and inserted pHS-cotG-KERQ7-M2 mutant plasmid, E. coli DH5α was transformed, and colony PCR verification was performed with P1-F and P1-R (PCR conditions were the same as KERQ7 amplification conditions). After verification, B. subtilis DB403 was transformed, and the transformants were picked again for colony PCR and fragment sequencing verification. The verified recombinant Bacillus subtilis were named B. subtilis DB403 pHS-cotG-KERQ7-M2, and stored at -80°C after expansion.

[0077] D: The molecular dynamics simulations involved in this invention all adopt the following steps: The protonation states of Glu and Asp in the protein structure are predicted using the PDB 2QR online server (https: / / server.poisonboltzmann.org / pdb2pqr), and the file is written to assign charges. The protein is confined in a well filled with TIP 3P water molecules, Na + and Cl - The system was kept in equilibrium in a 12-μm cubic box. The system was subjected to 10,000 steps (0.002 ps) and then equilibrated under the NVT system for 250,000 steps (0.002 ps per step) by increasing the temperature from 0 K to 300 K. Finally, the simulation was performed under the NVT system, controlled to run a 50 ns dynamics simulation at 300 K and 1 bar. Periodic boundary conditions were used. The calculation of electrostatic interactions used the particle mesh Ewald (PME) method. Trajectories and energies were calculated every 500 steps.

[0078] (2) During the implementation of the present invention, all primer sequences involved are shown in the following table:

[0079] Table 1 Primer sequences

[0080] Primer Name Primer Sequence Q7-F 5’-ggactagtgctcagacagtgccgtatgg-3’ Q7-R 5’-cccaagcttttaattgctcgccgcctgga-3’ P1-F 5’-gttggtagctcttgatccgg-3’ P1-R 5’-gttgctgttcctgttctgaat-3’ 64E-F 5’-ccgaatgcgacacaagattttcaatcagaggcacaacatgttgc-3’ 64E-R 5’-gcaacatgttgtgcctctgattgaaaatcttgtgtcgcattcgg-3’ 145E-F 5’-ttaaaaatgcggtggatacagcgaataatgagggcgttgttgttgttgcag-3’ 145E-R 5’-ctgcaacaacaacaacgccctcattattcgctgtatccaccgcatt-3’ 145D-F 5’-ttaaaaatgcggtggatacagcgaataatgatggcgttgttgttgttgcag-3’ 145D-R 5’-ctgcaacaacaacaacgccatcattattcgctgtatccaccgcatttt-3’ 98E-F 5’-gcctgtatgcggtgaaagttcttgatgagaatggagatggccaat-3’ 98E-R 5’-ccatgaatattggccatctccattctcatcaagaactttcaccgcatacag-3’ 36E-F 5’-gcattgaggcagcacatccggatctgaatgttgcagga-3’ 36E-R 5’-ggatgtgctgcctcaatgcctgtatccagaactgccac-3’ 17E-F 5’-ggcagtggaggcgcaaggatataaaggagcaaatgt-3’ 17E-R 5’-ccttgcgcctccactgccggatccttaatctgttcctg-3’

[0081] Example 1: Effect of mutation of the catalytic residues in the catalytic center of KERQ7 on the degradation activity of feather powder

[0082] (1) The present invention first selects keratinase KERQ7 (sequence number: KP694221) displayed on the surface of anchor protein cotG, which is denoted as cotG-KERQ7, wherein the nucleotide sequence of the mature fragment of KERQ7 is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2; wherein the steps of obtaining cotG-KERQ7 are as follows: using the sequence of the synthesized KERQ7 SEQ ID NO.1 as a template, amplifying KERQ7 with primers Q7-F and Q7-R (conditions: 94°C 3min, 94°C 30s, 55°C 30s, 72°C 1min, 30 cycles), and then inserting KERQ7 into the cotG plasmid using two endonucleases, Spe I and Hind III, to construct a recombinant plasmid, which is denoted as cotG-KERQ7;

[0083] (2) integrating the cotG-KERQ7 described in step (1) into the Bacillus subtilis shuttle expression vector pHS to form a shuttle plasmid pHS-cotG-KERQ7; then using the shuttle plasmid pHS-cotG-KERQ7 as a template, amplifying the pHS-cotG-KERQ7 plasmid using primers 64E-F and 64E-R by one-step PCR to finally obtain a mutant plasmid, which is recorded as pHS-cotG-KERQ7-64E;

[0084] The PCR amplification conditions were: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, and 30 cycles; the sequence of 64E-F was 5'-ccgaatgcgacacaagattttcaatca gag gcacaacatgttgc-3'; the sequence of 64E-R is 5'-gcaacatgttgtgc ctc tgattgaaaatcttgtgtcgcattcgg-3'.

[0085] (2) using the shuttle plasmid pHS-cotG-KERQ7 described in step (2) as a template, performing one-step whole plasmid PCR site-directed mutagenesis to change the catalytic residue His64 to Glu64, thereby obtaining a mutant plasmid pHS-cotG-KERQ7-64E, denoted as pHS-cotG-KERQ7-64E, wherein the KERQ7-64E keratinase mutant fragment in the mutant plasmid pHS-cotG-KERQ7-64E is denoted as KERQ7-64E; the nucleotide sequence of KERQ7-64E is shown in SEQ ID NO.3, and the amino acid sequence encoded by it is shown in SEQ ID NO.4; finally, pHS-cotG-KERQ7-64E is transformed into Escherichia coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-64E is obtained by extraction;

[0086] (3) In this example, the optimal pH of KERQ7 displayed on the surface of the capsid protein cotG of Bacillus subtilis spores and the catalytic center base catalytic residue mutant KERQ7-64E were compared. The complete plasmids of DH5α-pHS-cotG-KERQ7 and DH5α-pHS-cotG-KERQ7-64E were transformed into Bacillus subtilis DB403, respectively, to obtain the engineered bacteria B. subtilis DB403 pHS-cotG-KERQ7 and B. subtilis DB403pHS-cotG-KERQ7-64E that can be used to display keratinase on the surface of spores;

[0087] B.subtilis DB403 pHS-cotG-KERQ7 and B.subtilis DB403 pHS-cotG-KERQ7-64E were inoculated into LB medium, and cultured at 37°C and 200 rpm overnight (12 hours) to obtain a culture solution; the culture solution was then inoculated into DSM liquid medium at an inoculum amount of 1%, and cultured at 37°C and 200 rpm for 36 hours, and then the bacterial precipitate was collected by centrifugation, and the precipitate was resuspended in GTE Buffer containing 2 mg / mL lysozyme to obtain a resuspended solution, and then the precipitate was collected by centrifugation after incubation and washed with PBS Buffer, and the precipitate was collected after washing and resuspended in PBS Buffer again to obtain a recombinant spore protein suspension with keratinase displayed on the surface; the recombinant spore protein suspension or solid powder was keratinase preparation, which was respectively recorded as B.subtilis cotG-KERQ7 (abbreviated as wild) and B.subtilis cotG-KERQ7-64E (abbreviated as 64E). The components of GTE Buffer are: 10mM EDTA, 20mM Tris-HCl, pH=7.5, 50mM Glucose. The pH of PBS Buffer is 7.4, and the components of PBS Buffer are (based on 1L): 8g NaCl, 0.2g KCl, 1.42g Na2HPO4, 0.27g KH2PO4; the OD of the recombinant spore protein suspension is 600nm is 1.

[0088] The feather powder degradation activities of B. subtilis cotG-KERQ7 and B. subtilis cotG-KERQ7-64E keratinase preparations were determined and compared at pH 4, 5, and 6. The two enzyme preparations were diluted and plated to control the spore count to 1×10 8 spore suspension of B. subtilis cotG-KERQ7 and B. subtilis cotG-KERQ7-64E was added to phosphate buffer, and then feather powder was added as substrate. After constant temperature reaction, trichloroacetic acid (TCA) was added to terminate the reaction. After centrifugation, the supernatant was taken to measure OD 280nmThe control group was treated with TCA before the isothermal reaction, and the other operations were the same; the 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; the dosage relationship of the spore suspension, phosphate buffer, feather powder and trichloroacetic acid was 1.0 mL: 2.0 mL: 10 mg: 2.0 mL; the concentration of trichloroacetic acid was 10% (m / v); the isothermal reaction was carried out in a 37° C. isothermal shaker for 1 hour; the centrifugal conditions were 4° C. and 10,000 rpm for 10 minutes. The components of the phosphate buffer with a pH of 4 are: 7.71 mL of 0.2M Na2HPO4 and 12.29 mL of 0.1M citric acid solution; the components of the phosphate buffer with a pH of 5 are: 10.3 mL of 0.2M Na2HPO4 and 9.7 mL of 0.1M citric acid solution; the components of the phosphate buffer with a pH of 6 are: 12.63 mL of 0.2M Na2HPO4 and 7.37 mL of 0.1M citric acid solution.

[0089] Figure 1 (a) is the interaction near the catalytic domain, the blue dashed line represents hydrogen bond, and the gray dashed line represents van der Waals force; (b) is a schematic diagram of the hydrogen bond between the catalytic center and the substrate AAPF; (c) is the RMSD diagram of the molecular dynamics simulation of the 64E-AAPF complex simulated at different pH; (d) is the RMSF diagram of the molecular dynamics simulation of the 64E-AAPF complex simulated at different pH; (e) is the enzyme activity of wild-type KERQ7 and mutant 64E spore preparations.

[0090] The results are as follows Figure 1 As shown in Figure 6, the optimal pH of the 64E mutant shifted from 7 to 5, but the optimal pH activity did not increase significantly. In this example, His64 in the KERQ7 catalytic triad was mutated to glutamic acid by site-directed mutagenesis. In this mutant, although the original base catalytic residue His was lost, Glu could serve as a base catalytic residue and in acidic solution (H + ) environment, thereby robbing the proton on the proton donor Ser, thereby maintaining Ser's vigorous nucleophilic attack ability. The hydrogen bond between Ser221 and Glu64 also proves that glutamate must be protonated. This can explain why KERQ7-64E still has catalytic activity in an acidic environment.

[0091] On the other hand, molecular dynamics (MD) simulations of 64E with the keratin analog AAPF at different pH conditions for 40 ns showed that the typical catalytic triad became a catalytic dyad of Ser-Glu. Ser221 was covalently linked to the substrate amide bond and remained active in the experiment, indicating that Ser221 is still a nucleophilic residue. The distance between Glu64 and Ser221 is Within 100%, hydrogen bonds are formed between Glu64 and Val68, and the van der Waals forces between Glu64 and Tyr217 help to fix Glu64, bury it in the active site, and reduce the pKa of Glu64 (from 6.15 to 4.67). In this example, the basic catalytic residues in the catalytic triad of KERQ7 were mutated, and the resulting catalytic dyad still has protein hydrolysis activity. In particular, acidic glutamic acid acts as a basic catalytic residue in this round of mutations, reflecting its potential to enhance activity in an acidic environment.

[0092] Conclusion: The results of Example 1 show that although the optimal pH of KERQ7-64E shifts toward the acidic side, the activity has not been significantly improved and the acid resistance needs to be strengthened. Therefore, the mutant KERQ7-64E should be modified to remove unstable and positively charged amino acids on the enzyme protein surface.

[0093] Example 2: Effect of mutation of unstable positively charged residues on the degradation activity of feather powder in 64E

[0094] In this example, the surface amino acids of the mutant KERQ7-64E in Example 1 were modified to obtain the mutant Q7-M1.

[0095] (1) The amino acid sequence of the mutant fragment KERQ7-64E was used as a template and Alphafold2 was selected to construct a three-dimensional protein model. The resulting protein model was recorded as Q7-64E. Subsequently, Q7-64E was molecularly docked with the keratin analog AAPF (N-succinyl-ala-ala-pro-phe-p-nitroanilide, PubChem CID 5496888) to obtain a Q7-64E-AAPF protein-ligand complex. The complex was subjected to molecular dynamics simulation in an acidic pH environment (pH = 4-6). The root mean square fluctuation (RMSF) values ​​of amino acids at each site of the protein three-dimensional structure were different under different pH environments. The stability of the protein was evaluated by the height of the RMSD trend line, and the lower the RMSD, the more stable it was. Therefore, the protein three-dimensional structure model whose root mean square deviation (RMSD) change tended to a stable equilibrium was selected and recorded as 64E (i.e., the pH environment was 5). The trajectory files obtained by molecular dynamics simulation of Q7-64E-AAPF, including root mean square deviation (RMSD), root mean square fluctuation (RMSF) and hydrogen bonds were analyzed by VMD 1.9.3, and the static structure of Q7-64E-AAPF and the visualization of its function were displayed by Pymol 2.5.5.

[0096] (2) Based on the root mean square fluctuation (RMSF) values ​​of each amino acid site of model 64E as a reference, the modification sites were selected; the amino acids located on the surface of the enzyme protein that were unstable (large RMSF changes) and positively charged were replaced. The modification sites included surface amino acids Arg145, Arg98, His36, and His17;

[0097] S1. Using DH5α-pHS-cotG-KERQ7-64E as a template, PCR site-directed mutagenesis was performed. Primers 145E-F and 145E-R were used to amplify the DH5α-pHS-cotG-KERQ7-64E plasmid by one-step PCR, and the surface amino acid Arg145 was changed to Glu145 to obtain the mutant plasmid pHS-cotG-KERQ7-145E, recorded as pHS-cotG-KERQ7-145E. Then, pHS-cotG-KERQ7-145E was transformed into Escherichia coli DH5α, and the complete DH5α-pHS-cotG-KERQ7-145E plasmid was extracted.

[0098] The PCR amplification conditions were as follows: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min for 30 cycles;

[0099] The primer sequence for 145E-F is 5'-ttaaaaatgcggtggatacagcgaataat gag ggcgttgttgttgttgttgcag-3'; the primer sequence for 145E-R is 5'-ctgcaacaacaacaacgcc ctc attattcgctgtatccaccgcatt-3'.

[0100] Using DH5α-pHS-cotG-KERQ7-64E as a template, PCR site-directed mutagenesis was performed. Primers 145D-F and 145D-R were used to amplify the DH5α-pHS-cotG-KERQ7-64E plasmid by one-step PCR, and the surface amino acid Arg145 was changed to Asp145 to obtain the mutant plasmid pHS-cotG-KERQ7-145D, recorded as pHS-cotG-KERQ7-145D. Then pHS-cotG-KERQ7-145D was transformed into Escherichia coli DH5α, and the complete DH5α-pHS-cotG-KERQ7-145D plasmid was extracted.

[0101] The PCR amplification conditions were as follows: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min for 30 cycles;

[0102] The primer sequence for 145D-F is 5'-ttaaaaatgcggtggatacagcgaataat gat ggcgttgttgttgttgttgcag-3'; the primer sequence of 145D-R is 5'-ctgcaacaacaacaacgcc atc attattcgctgtatccaccgcatttt-3'.

[0103] S2. Using DH5α-pHS-cotG-KERQ7-145E obtained in S1 as a template, perform PCR site-directed mutagenesis. Use primers 98E-F and 98E-R to amplify the DH5α-pHS-cotG-KERQ7-145E plasmid by one-step PCR. Change the surface amino acid Arg98 to Glu98 to obtain the mutant plasmid pHS-cotG-KERQ7-98E, recorded as pHS-cotG-KERQ7-98E. Transform pHS-cotG-KERQ7-98E into Escherichia coli DH5α, and extract to obtain the complete plasmid DH5α-pHS-cotG-KERQ7-98E.

[0104] The conditions for PCR amplification were: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, and 30 cycles; the primer sequence for 98E-F was: 5'-gcctgtatgcggtgaaagttcttgat gag aatggagatggccaat-3'; the primer sequence of 98E-R is: 5'-ccatgaatattggccatctccatt ctc atcaagaactttcaccgcatacag-3'.

[0105] S3. Using DH5α-pHS-cotG-KERQ7-98E obtained in S2 as a template, perform one-step whole-plasmid PCR site-directed mutagenesis. Use primers 36E-F and 36E-R to amplify the DH5α-pHS-cotG-KERQ7-98E plasmid by one-step PCR, change the surface amino acid His36 to Glu36, and obtain the mutant plasmid pHS-cotG-KERQ7-36E, recorded as pHS-cotG-KERQ7-36E. Transform pHS-cotG-KERQ7-36E into Escherichia coli DH5α, and extract to obtain the complete plasmid DH5α-pHS-cotG-KERQ7-36E.

[0106] The conditions for PCR amplification were: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min for 30 cycles;

[0107] The sequence of 36E-F is: 5'-gcatt gag gcagcacatccggatctgaatgttgcagga-3'; the sequence of 36E-R is: 5'-ggatgtgctgc ctc aatgcctgtatccagaactgccac-3'.

[0108] S4. Using DH5α-pHS-cotG-KERQ7-36E obtained in S3 as a template, performing one-step whole-plasmid PCR site-directed mutagenesis to change the surface amino acid His17 to Glu17, obtaining a mutant plasmid pHS-cotG-KERQ7-17E, denoted as pHS-cotG-KERQ7-M1, transforming pHS-cotG-KERQ7-M1 into Escherichia coli DH5α, extracting to obtain the complete plasmid DH5α-pHS-cotG-KERQ7-M1, wherein the KERQ7 keratinase mutant fragment is denoted as Q7-M1, the nucleotide sequence of Q7-M1 is shown in SEQ ID NO.5, and the amino acid sequence encoded by it is shown in SEQ ID NO.6;

[0109] The conditions for PCR amplification were: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min for 30 cycles;

[0110] The sequence of 17E-F is: 5'-ggcagtg gag gcgcaaggatataaaggagcaaatgt-3'; the sequence of 17E-R is: 5'-ccttgcgc ctc cactgccggatccttaatctgttcctg-3'.

[0111] S5. The recombinant plasmids DH5α-pHS-cotG-KERQ7-64E, DH5α-pHS-cotG-KERQ7-145E, DH5α-pHS-cotG-KERQ7-145D, DH5α-pHS-cotG-KERQ7-98E, DH5α-pHS-cotG-KERQ7-36E and DH5α-pHS-cotG-KERQ7-M1 obtained in steps S1-S4 were verified by colony PCR using P1-F and P1-R, respectively (PCR amplification conditions: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, and 30 cycles); after verification, they were transformed into B. subtilis DB403, and the transformants were picked again for colony PCR and fragment sequencing verification, and the verified recombinant Bacillus subtilis were named B. subtilis DB403pHS-cotG-KERQ7-64E (abbreviated as 64E), B. subtilis DB403pHS-cotG-KERQ7-145E (abbreviated as 145E), B. subtilis DB403 pHS-cotG-KERQ7-145D (abbreviated as 145D), B. subtilis DB403pHS-cotG-KERQ7-98E (abbreviated as 98E), B. subtilis DB403 pHS-cotG-KERQ7-36E (abbreviated as 36E) and B. subtilis DB403 pHS-cotG-KERQ7-M1 (abbreviated as M1).

[0112] (3) Using the amino acid sequence of the mutant fragment Q7-M1 of keratinase in S3 as a template, Alphafold2 was selected to construct a three-dimensional protein model, and the resulting protein model was recorded as KERQ7-M1. Subsequently, KERQ7-M1 was molecularly docked with the keratin analog AAPF (N-succinyl-ala-ala-pro-phe-p-nitroanilide, PubChem CID 5496888), and the docking posture selected the conformation in which the catalytic amino acid Ser221 of KERQ7-M1 directly interacted with the amide bond of AAPF to obtain the KERQ7-M1-AAPF protein ligand complex. The complex was subjected to molecular dynamics simulation in an acidic pH environment, and the acidic pH environment in the molecular dynamics simulation was 4, 5, and 6. Through the results of this dynamics simulation, the area on the enzyme protein surface that was greatly affected by the acidic pH environment was obtained. After simulating the KERQ7-M1-AAPF protein ligand complex in an acidic pH environment, the protein three-dimensional structure model was affected by different environmental pH under different pH environments, and the root mean square fluctuation (RMSF) of the amino acids at each site was significantly affected by the acidic pH environment. The change of RMSF was used as the standard to evaluate the impact of acidic environment.

[0113] This example introduces the process of mutation from mutant KERQ7-64E to Q7-M1, and compares the optimal pH of mutant B. subtilis cotG-KERQ7-64E displayed on the surface of capsid protein cotG on the surface of Bacillus subtilis spores in Example 1 with B. subtilis cotG-KERQ7-M1 after mutation of unstable positively charged residues on the surface of enzyme protein;

[0114] The spore enzyme preparations B. subtilis cotG-KERQ7-64E and B. subtiliscotG-KERQ7-M1 were prepared according to the method in Example 1, except that the complete plasmids of DH5α-pHS-cotG-KERQ7 and DH5α-pHS-cotG-KERQ7-64E were replaced with DH5α-pHS-cotG-KERQ7-64E and DH5α-pHS-cotG-KERQ7-M1 respectively, and the subsequent steps were the same, and finally B. subtilis cotG-KERQ7-64E and B. subtilis cotG-KERQ7-M1 were obtained, and the feather powder degradation activity changes of B. subtilis cotG-KERQ7-64E and B. subtilis cotG-KERQ7-M1 in different pH (4-6) environments were compared using the feather powder degradation activity method in Example 1.

[0115] The final mutant 64E has an optimal enzymatic pH of about 5, and its stability is evaluated by the RMSF value. Residues with high RMSF values ​​are generally considered to be unstable under the set conditions. Therefore, in this example, the potential mutation sites are predicted through the RMSF simulation results at pH = 5, and the positively charged amino acids with the largest surface deviation of the protein, that is, the most unstable, are selected for modification. Finally, the results of the structure and trajectory analysis after molecular dynamics simulation are compared, and it is predicted that 98E, 36E, and 17E are the key amino acids that are most affected by the acidic environment (RMSF>0.8), and their RMSF values ​​are 98E=1.1999, 36E=0.8946, and 17E=0.8778, respectively. Considering that the strategy of this example is activity-stability trade-off + surface charge replacement, in order to determine the specific gain effects of glutamate and aspartic acid, the relatively stable 145R (RMSF=0.5376) located at the end of the α-helix is ​​first selected for site-directed mutagenesis. From Figure 2 It can be seen from a that the mutation of 145E is higher than that of 145D. This may be because glutamic acid has a larger side chain conformation than aspartic acid, which makes it more stable in space, making it less vulnerable to attack. Using the online binding energy prediction tool DUET ( DUET(uq.edu.au) ) predicted that the ΔΔG after E145D mutation is -0.325 kcal / mol. The higher folded conformational entropy stabilizes the protein, indicating that the mutation 145E at this point is more stable than 145D. Based on this result, the mutation of glutamic acid was used for the subsequent mutation of other sites in this example. Then, Arg 98 (RMSF = 1.1999), which has the largest RMSF deviation and is the most unstable, was mutated. Figure 2 As can be seen in Figure (a), replacing negatively charged amino acids in unstable regions can improve activity in acidic environments, and the activity at pH = 5 is increased to 202.85±2.214% of B. subtilis cotG-KERQ7-64E. After mutating several unstable (RMSF>0.8) surface positively charged amino acids, the enzyme preparation B. subtilis cotG-KERQ7-M1 was finally obtained;

[0116] The enzyme activity at pH = 4 was increased to 272.72 ± 2.584% of that of B. subtilis cotG-KERQ7-64E. The mutant was then subjected to bioinformatics analysis. To reveal the potential mechanism of the improved acid tolerance of M1 after the introduction of the mutation, pH-based molecular dynamics simulation was used to compare the structural stability of 64E and M1. Figure 2 As shown in Figure (b), the average backbone RMSD values ​​of M1 at pH = 4, pH = 5, and pH = 6 are Lower than the RMSD value of Q7-WT ( and ), the lower RMSD value indicates that the overall structure of M1 is more stable under acidic conditions, which may lead to the improvement of its acid tolerance.

[0117] The molecular dynamics simulations of 64E at pH = 5 and M1 at pH = 4 were simulated for a long time until the equilibrium structure was derived, and the two were analyzed and compared. First of all, considering that the theme of this round of mutation is the replacement of surface charge, the charge change on the surface of the protein must lead to the difference in the dissociation state and protonation state of the relevant groups. Therefore, the structure of the protein is affected by the environmental pH, resulting in different changes in its conformation and folding state. Using the protein plus online server ( https: / / proteins.plus / ), the substrate binding pocket volumes of 64E and M1 were calculated, and M1 has a deeper substrate binding pocket and larger pocket capacity The bottom of the pocket is the catalytic residue Ser221, which is the S1 substrate binding pocket; the larger and deeper S1 pocket makes the binding to the substrate P1 site more stable, thus leading to an increase in keratin degradation activity. From the analysis of the cavity inside the S1 binding pocket, the total formal charge in the M1 cavity is -1, which is higher than 64E, so it is more adaptable to acidic environments and has a better catalytic effect under acidic conditions ( Figure 2 (c)).

[0118] In the previous round of mutation, the posture of the enzyme-substrate catalytic reaction and the tone of the acid-resistant mutation were determined, and then the molecular dynamics simulation of the entire protein-ligand complex system at different pH in an acidic environment was carried out. By mutating the unstable, positively charged amino acid on the surface into acidic glutamic acid, the essence is to increase the amount of negative charge on the enzyme surface to improve the enzyme's adaptability to the acidic environment under the premise of determining the catalytic mode and the binding posture of the active site. As the overall protonation state of the enzyme surface and the dissociation state of each residue become more and more adapted to the acidic environment, the conformation of the substrate binding pocket will become deeper and larger, and then the binding conformation of the enzyme and substrate will become more stable, thereby improving the activity and acid resistance in an acidic environment and achieving a balance between activity and stability. From the experimental results and bioinformatics analysis, this strategy is feasible. The idea of ​​changes in the volume of the substrate binding pocket and the electrostatic potential inside the cavity caused by changes in the surface electrostatic potential can also provide a reference for the subsequent modification of the acid resistance of other serine proteases.

[0119] Conclusion: The results of Example 2 show that although the acid resistance of M1 has been significantly improved, the enzyme activity is still insufficient. Therefore, the surface flexibility of the enzyme protein of mutant M1 should be improved to make it more adaptable to acidic environments.

[0120] Example 3: Effect of adding “flexible tentacles” to the surface of M1 on the degradation activity of feather powder

[0121] In this example, mutant Q7-M2 was obtained by inserting a repeating fragment into mutant Q7-M1 in Example 2. Combined with the RMSF results of molecular dynamics simulation of KERQ7-M1-AAPF in an environment between pH 4 and 6, the region outside the catalytic pocket of KERQ7-M1 that is most affected by changes in the acidic environment, that is, the region with the largest change in RMSF value (Glu36-Asn43) was selected; based on the selected region Glu36-Asn43, Glu36 was then used as the insertion site, and the AAEPVLDTGIE amino acid fragment was inserted after the Glu36 site of the keratinase mutant KERQ7-M1 on the pHS-cotG-KERQ7-M1 plasmid to obtain a complete mutant plasmid with the new insertion fragment, which was recorded as pHS-cotG-KERQ7-M2; and pHS-cotG-KERQ7-M2 was transformed into Escherichia coli DH5α, and the DH5α-pHS-cotG-KERQ7-M2 complete plasmid was extracted, wherein the KERQ7 keratinase mutant fragment was recorded as Q7-M2, and the nucleotide sequence of Q7-M2 is as shown in SEQ ID NO: 1. NO.7, and the amino acid sequence encoded by it is shown in SEQ ID NO.8; pHS-cotG-KERQ7-M2 is transformed into Escherichia coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-M2 is extracted, wherein the KERQ7 keratinase mutant fragment is recorded as Q7-M2, the nucleotide sequence of Q7-M2 is shown in SEQ ID NO.7, and the amino acid sequence encoded by it is shown in SEQ ID NO.8;

[0122] Using the amino acid sequence of the keratinase mutant fragment KERQ7-64E as a template, Alphafold2 was selected to construct a three-dimensional protein model, and the resulting protein model was recorded as Q7-64E; subsequently, Q7-64E and the keratin analog AAPF (N-succinyl-ala-ala-pro-phe-p-nitroanilide, PubChem CID 5496888) were molecularly docked using Autodock, and the docking posture should select a conformation in which the catalytic amino acid Ser221 of M2 directly interacts with the AAPF amide bond to obtain a Q7-64E-AAPF protein ligand complex. The complex was subjected to molecular dynamics simulation in an acidic pH environment (pH = 4-6), and the protein three-dimensional structure model whose root mean square deviation (RMSD) change tended to a stable equilibrium was selected as M2 (i.e., the pH environment was 5.5). The trajectory files obtained by molecular dynamics simulation of M2-AAPF, including root mean square deviation (RMSD), root mean square fluctuation (RMSF), number of hydrogen bonds, and interatomic distances were analyzed using VMD 1.9.3, and the static structure of M2-AAPF and the visualization of its function were displayed by Pymol 2.5.5.

[0123] The complete plasmid DH5α-pHS-cotG-KERQ7-M2 was transformed into Bacillus subtilis DB403 to obtain the engineered bacteria B. subtilis DB403 pHS-cotG-KERQ7-M2 that can be used to display keratinase on the surface of spores. The B. subtilis DB403 pHS-cotG-KERQ7-M2 engineered bacteria was inoculated into LB medium and cultured overnight at 37°C and 200 rpm to obtain a culture solution. The culture solution was then inoculated into DSM liquid medium, and the bacterial precipitate was collected by centrifugation after 37°C and 200 rpm for 36 hours. The precipitate was resuspended in GTE Buffer containing lysozyme to obtain a resuspension. After incubation (37°C, 60 minutes), the precipitate was collected by centrifugation and washed with PBS Buffer. After washing, the precipitate was collected and resuspended in PBS again. Buffer, to obtain a recombinant spore protein suspension with keratinase displayed on the surface; or collect the precipitate after washing and dry it at low temperature to obtain a solid powder; the recombinant spore protein suspension or solid powder is a keratinase preparation, recorded as B.subtilis cotG-KERQ7-M2 (abbreviated as M2). The concentration of lysozyme in GTE Buffer is 2 mg / mL, and the components of GTE Buffer are: 10 mM EDTA, 20 mM, pH=7.5 Tris-HCl, 50 mM Glucose. The pH of PBS Buffer is 7.4, and the components of PBS Buffer are (based on 1L): 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4; the OD of the recombinant spore protein suspension is 600nm is 1.

[0124] This example introduces the selection process of the mutation region of mutant Q7-M2, compares the optimal pH of the mutant B. subtilis cotG-KERQ7-M1 displayed on the surface of the capsid protein cotG on the surface of Bacillus subtilis spores in Example 2 and B. subtilis cotG-KERQ7-M2 after the surface "flexible tentacles" are increased, and compares the acid stability of B. subtilis cotG-KERQ7-M2 and B. subtilis cotG-KERQ7 displayed on the surface of the capsid protein cotG on the surface of Bacillus subtilis spores in Example 1.

[0125] The spore enzyme preparations B. subtilis cotG-KERQ7-M1 and B. subtiliscotG-KERQ7-M2 were prepared according to the method in Example 1, except that the complete plasmids of DH5α-pHS-cotG-KERQ7 and DH5α-pHS-cotG-KERQ7-64E were replaced with DH5α-pHS-cotG-KERQ7-M1 and DH5α-pHS-cotG-KERQ7-M2, and the remaining steps were the same, and finally B. subtilis cotG-KERQ7-M1 (abbreviated as M1) and B. subtilis cotG-KERQ7-M2 (abbreviated as M2) were obtained;

[0126] The feather powder degradation activity of B. subtilis cotG-KERQ7-M1 and B. subtiliscotG-KERQ7-M2 in different pH (4-6) environments was compared using the feather powder degradation activity method in Example 1. Acid stability The residual enzyme activity of B. subtiliscotG-KERQ7-M2 (abbreviated as M2) and B. subtilis cotG-KERQ7 (abbreviated as Wild-type) after incubation in buffers of different acidic pH (pH = 4 / 4.5 / 5 / 5.5 / 6 / 6.5) at room temperature for 1h and 3h was investigated, and the enzyme activity incubated for 0h in each reaction environment was taken as 100%.

[0127] From the previous round of experiments and molecular dynamics simulation results, it can be seen that although the adaptability of M1 to the acidic solution environment is improving with the accumulation of negative charges on the surface, as the number of protonated amino acids on the surface increases, the electrostatic interaction within the protein may be enhanced, causing the M1 molecules to fold and bind more tightly and become rigid. Therefore, despite the subsequent mutation of the positive charge on its surface, the activity has not been significantly improved. It seems that M1 needs to restore a certain flexibility in order to improve its catalytic activity in acidic environments.

[0128] Based on this idea, the mutation selection in Example 3 introduces a new flexible region in M1 to improve the catalytic activity. Through the RMSF results of molecular dynamics simulation of M1, the region around the substrate binding S1 pocket with the largest change between pH4 and 6 was selected. It was preliminarily determined that it had an effect on the activity of keratinase, and this region was modified. The five amino acids with high flexibility in region 1 (GIEAA, RMSF of 0.7609, 0.7406, 0.9605, 1.0583, and 0.9004, all higher than the average RMSF) were expressed repeatedly so that they could form a large flexible ring region ( Figure 3After site-directed mutagenesis of this structure, spore enzyme preparations were collected to test enzyme activity. It was found that although its optimal pH shifted from 4-5 of B. subtilis cotG-KERQ7-M1 to about 5.5, it increased to 435% of 64E in a pH = 5 environment ( Figure 4 ), which was significantly improved, and the acid tolerance of B. subtilis cotG-KERQ7-M2 was subsequently evaluated.

[0129] from Figure 5 It can be seen that the activity and acid stability of B.subtilis cotG-KERQ7-M2 were significantly improved after three rounds of mutation. Among them, B.subtilis cotG-KERQ7-M2 still had 96.67% and 97.308% of activity after incubation at pH = 5 and 5.5 for 1h, and the residual activity of other acidic pH and time gradients was also improved, achieving substantial results. Therefore, it is basically determined that the addition of the flexible loop region has a benefit on activity and acid resistance, and the acid resistance of the spore-displaying keratinase mutant B.subtilis cotG-KERQ7-M2 has indeed been improved. The following is a bioinformatics verification and analysis.

[0130] Considering whether the addition of flexible regions would affect the enzyme function and effect, the three-dimensional structure of the sequence was predicted and evaluated, and then molecular docking was performed to find its key docking posture. The M2-AAPF and M1-AAPF systems were simulated with different pH values ​​for 50ns, and the protein-substrate binding mode was observed. Figure 6 (a) shows the distance between the catalytic Ser hydroxyl oxygen skeleton and the carbonyl oxygen of the substrate AAPF. It can be seen that after the protein structure is dynamically stabilized, the distance between the atoms of the M2-AAPF interaction remains at Around, smaller than M1-AAPF The shorter interaction distance may reflect a stronger binding ability, which is why the activity of M2 is improved. The equilibrium structure of the protein-substrate complex after 50ns molecular dynamics simulation was derived and displayed using Pymol. The catalytic dyad 75Glu-232Ser formed a hydrogen bond with the substrate AAPF amide group and Figure 6 As shown in (b), at pH = 5.5, the hydrogen bond formed between the catalytic residue 232Ser and the amide group on the substrate AAPF has existed since the structure stabilized, indicating that it has been stably following the serine catalytic mode of action, attacking the substrate with nucleophilicity, and the mode of action of M2 has not changed, and it is still a keratinase belonging to the serine protease family. In addition, 136Ser, which participates in the formation of the oxygen anion hole, also participates in the nucleophilic attack on the carbonyl oxygen of the substrate AAPF to form a hydrogen bond, and these hydrogen bonds form a regular tetrahedral cavity (Figure 6 b). Use the ParKVFinder Tools plug-in to calculate the cavity volume of the oxygen anion hole formed by Q7-64E and M2, as shown in Figure 6 In (c), the results show that M2 has a larger oxygen anion hole in terms of volume and surface area than Q7-64E. It seems that the tetrahedral cavity formed by several hydrogen bonds not only stabilizes the formation of the oxygen anion hole, but also increases the volume of the oxygen anion hole, thereby improving the nucleophilic attack ability of the catalytic site on the substrate, thereby greatly improving the enzyme activity. Figure 6 In (d), it can be seen that the local rigidity and flexibility of the modified part have changed greatly, the surface hydrophilicity has been greatly improved, and the catalytic residues are buried deep inside, while the accessible area of ​​the solution is increased (from Q7-64E To M2 ) may also have some impact on the activity enhancement. Compared with M1, M2 has not undergone any changes in the nature of the enzyme action, so the changes between the two were compared based on the catalytic principle of serine protease.

[0131] Hydrogen bonding is an important factor in maintaining the stability of protein structure. M2 has fewer solute-solute hydrogen bonds and more solute-solvent hydrogen bonds than M1. The Bridges in Table 2 represent solvent bridging interactions (i.e., two or more solute residues are hydrogen-bonded to a single solvent residue). These indicate that the protein-ligand complex of M2 has higher solubility, protein surface hydrophilicity, and adaptability to the simulated environment (pH = 5) than the M1 complex, so M2 is more flexible in this solution environment. The improvement in overall flexibility can also be seen from the RMSD diagram of the two. The M2 skeleton RMSD is higher than that of M1 ( Figure 6 e). This indicates that this round of mutations has successfully improved the flexibility of M2 in acidic solutions. Although the optimal pH has shifted toward neutrality, the activity has been improved. This indicates that the idea of ​​modifying enzymes based on the effects of protein rigidity, flexibility, and hydrophilicity on enzyme activity is also feasible.

[0132] Table 2 Total number of hydrogen bonds in the equilibrium structures of M2 and M1 enzyme proteins

[0133]

[0134] Example 4: Preparation of feather meal feed by bacterial enzyme synergistic fermentation;

[0135] The feed raw materials are composed of feather meal, bean dregs and second-rate flour, and the mass ratio of feather meal: bean dregs: second-rate flour is 4:3:3; the addition amounts of other substances are calculated as a mass percentage of the feed raw materials;

[0136] The total inoculation amount of the fermented seed liquid in the feed raw material is 6% (ml / g), and the inoculation ratio is the volume ratio of cerevisiae: Bacillus licheniformis: Lactobacillus fermentum: Lactobacillus rhamnosus = 2:2:1:1. In addition, in order to promote the amino acid balance of the feather meal feed, 0.25% Met and 0.25% Lys are additionally added to form a fermentation system; the water content is controlled to be 45%, and the fermentation conditions are: placed in a constant temperature incubator at a temperature of 35°C for anaerobic fermentation for 7 days.

[0137] The following 6 groups were set up, with three parallels in each group, and the pH, small peptide and free amino acid content of each group were measured.

[0138] 1. The raw materials were mixed to form a fermentation system but no fermentation was performed, which is the unfermentation group;

[0139] 2. The raw materials are mixed to form a fermentation system, and fermentation is carried out according to the above operation, which is recorded as the fermentation group (Fermentation + mixed fermentation strain)

[0140] 3. The raw materials were mixed to form a fermentation system, and 10% B. subtilis DB403 pHS-cotG spore enzyme preparation (mL / g) was added, which was recorded as cotG enzyme preparation fermentation group (Fermentation+cotG);

[0141] 4. The raw materials were mixed to form a fermentation system, and 10% B. subtilis pHS-cotG-KERQ7 spore enzyme preparation (mL / g) was added, which was recorded as cotG-kerQ7 enzyme preparation fermentation group (Fermentation+cotG-KERQ7);

[0142] 5. The raw materials were mixed to form a fermentation system, and 10% B. subtilis pHS-cotG-KERQ7-M2 spore enzyme preparation (mL / g) was added, which was recorded as 10% cotG-M2 enzyme preparation fermentation group (Fermentation+10% cotG-M2);

[0143] 6. The raw materials were mixed to form a fermentation system, and 12% B. subtilis pHS-cotG-KERQ7-M2 spore enzyme preparation (mL / g) was added, which was recorded as 12% cotG-M2 enzyme preparation fermentation group (Fermentation + 12% cotG-M2)

[0144] 7. The raw materials were mixed to form a fermentation system, and 15% B. subtilis pHS-cotG-KERQ7-M2 spore enzyme preparation (mL / g) was added, which was recorded as 15% cotG-M2 enzyme preparation fermentation group (Fermentation + 15% cotG-M2)

[0145] Table 3 Data table of the effect of spore enzyme preparation on the content of small peptides produced by feather powder fermented feed

[0146]

[0147] The results showed that the content of small peptides in unfermented feather meal feed was only 4.33±0.19%. After mixed fermentation with yeast, Bacillus licheniformis and lactic acid bacteria, the content of small peptides increased to 7.216±0.88. Different spore enzyme preparations were further added. After 7 days of fermentation, the small peptide contents of cotG empty spores, cotG-KERQ7, 10% cotG-M2, 12% cotG-M2 and 15% cotG-M2 were 7.65±0.37%, 12.69±0.52%, 17.72±0.64%, 19.31±0.21% and 22.25±0.79%, respectively. Figure 7 The effect of spore enzyme preparation on the content of small peptides produced by feather meal fermented feed.

[0148] Figure 8 This is the standard peak diagram of 17 acid-soluble amino acids separated by HPLC, which shows that the free amino acids in the sample can be separated well under this chromatographic condition. At the same time, the free amino acids of the above four groups of samples were determined, and the results are as follows Figure 9 As shown, the free amino acids of the four groups of samples were determined according to the grouping. The total free amino acid content of the cotG empty spore group was only 8.2±0.41 mg / g, but the total free amino acid content of each group after adding keratinase was greatly improved. The free amino acid contents of the wild-type KERQ7 spore enzyme preparation, 10% mutant M2 spore enzyme preparation, 12% mutant M2 spore enzyme preparation and 15% mutant M2 spore enzyme preparation were 13.54±0.122 mg / g, 23.93±0.43 mg / g, 30.45±0.28 mg / g and 37.3±0.49 mg / g, respectively. The change trend of the free amino acid content in each group was basically consistent with the small peptide content. From the experimental results, it can be seen that feather meal is rich in cysteine, valine, etc. The supplemented methionine and lysine also improve the nutritional value of feather meal feed. After fermentation, various amino acids are relatively balanced, and fermentation brings a significant increase in the content of flavor amino acids such as alanine, aspartic acid, and glutamic acid. While balancing the amino acids in the feed, it also brings about an improvement in the umami taste, providing a basis for the utilization of feather meal as feed.

[0149] Example 5: Spore-displaying keratinase solid powder is used for the preparation of feather meal feed by bacterial enzyme synergistic fermentation

[0150] Based on Example 4, the difference is that the suspension (B. subtilis cotG-KERQ7-M2) is replaced by a solid powder; that is, the keratinase preparation B. subtilis cotG-KERQ7-M2 is added to the fermentation system in the form of a freeze-dried solid powder, and the addition amount is 0.5%, and other conditions remain unchanged; the final measured small peptide content is 16.89±0.88%, and the total free amino acid content is 23.02±0.26 mg / g, which is not significantly different from the group in which 10% spore suspension enzyme preparation B. subtilis cotG-KERQ7-M2 is added in Example 4, indicating that the addition of the spore enzyme preparation in the form of a solid powder is equally efficient and feasible, and can achieve significant technical effects.

[0151] 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 skilled 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.

[0152]

[0153]

Claims

1. A KERQ7 keratinase mutant fragment, characterized in that: The KERQ7 keratinase mutant fragment is Q7-M2, the gene nucleotide sequence of Q7-M2 is shown in SEQ ID NO.7, and the amino acid sequence encoded by it is shown in SEQ ID NO.

8.

2. A plasmid vector carrying the KERQ7 keratinase mutant fragment according to claim 1.

3. A recombinant engineered bacterium carrying the gene according to claim 1 or the plasmid vector according to claim 2.

4. A method for preparing an acid-resistant high-activity keratinase preparation, characterized in that: The recombinant engineered bacteria according to claim 3 are inoculated into a fermentation medium for cultivation, and then the bacteria are collected by centrifugation and resuspended in a PBS buffer to obtain a recombinant spore protein suspension with keratinase displayed on the surface; or the bacteria are collected and subjected to low-temperature drying to obtain a solid powder; the recombinant spore protein suspension or solid powder is an acid-resistant high-activity keratinase preparation, which is recorded as B. subtilis cotG-KERQ7-M2.

5. The method for preparing an acid-resistant high-activity keratinase preparation according to claim 4, characterized in that: The following steps are involved: (1) The present invention first selects keratinase KERQ7 displayed on the surface of anchor protein cotG, denoted as cotG-KERQ7; wherein the nucleotide sequence of the mature fragment of KERQ7 is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2; (2) integrating the cotG-KERQ7 described in step (1) into the Bacillus subtilis shuttle expression vector pHS to form a shuttle plasmid pHS-cotG-KERQ7; (3) Using the shuttle plasmid pHS-cotG-KERQ7 described in step (2) as a template, performing one-step whole plasmid PCR site-directed mutagenesis to change the catalytic residue His64 to Glu64, thereby obtaining a mutant plasmid pHS-cotG-KERQ7-64E, which is recorded as pHS-cotG-KERQ7-64E, wherein the KERQ7-64E keratinase mutant fragment in the mutant plasmid pHS-cotG-KERQ7-64E is recorded as KERQ7-64E; the nucleotide sequence of KERQ7-64E is shown in SEQ ID NO.3, and the amino acid sequence encoded by it is shown in SEQ ID NO.4; finally, pHS-cotG-KERQ7-64E is transformed into Escherichia coli DH5α, and the DH5α-pHS-cotG-KERQ7-64E complete plasmid is extracted; (4) Using the amino acid sequence of the keratinase mutant fragment KERQ7-64E in step (3) as a template, a three-dimensional protein model is constructed, and the obtained protein model is recorded as Q7-64E; Q7-64E is then molecularly docked with the keratin analog AAPF to obtain a Q7-64E-AAPF protein ligand complex, and the complex is subjected to molecular dynamics simulation in an acidic pH environment. The unstable, positively charged amino acid sites located on the surface of the enzyme protein during the catalytic process can be obtained through the molecular dynamics simulation results; after the Q7-64E-AAPF protein ligand complex is simulated in an acidic pH environment, the root mean square deviation (RMSD) change trend of the protein three-dimensional structure model under different pH environments is different, and the root mean square fluctuation value of the amino acid at each site of the protein three-dimensional structure under different pH environments is different. The stability is evaluated by the height of the RMSD trend line, and the lower the RMSD, the more stable it is; Therefore, the protein three-dimensional structure model with the lowest root mean square deviation was selected and recorded as 64E; The modification sites were selected based on the root mean square fluctuation values ​​of each amino acid site of model 64E; The modification sites include surface amino acids Arg145, Arg98, His36 and His17; (5) Using DH5α-pHS-cotG-KERQ7-64E obtained in step (3) as a template, performing one-step whole plasmid PCR site-directed mutagenesis to change the surface amino acid Arg145 to Glu145, thereby obtaining a mutant plasmid pHS-cotG-KERQ7-145E, recorded as pHS-cotG-KERQ7-145E, and then transforming pHS-cotG-KERQ7-145E into Escherichia coli DH5α, and extracting to obtain the complete plasmid DH5α-pHS-cotG-KERQ7-145E; (6) Preparation of mutant plasmid pHS-cotG-KERQ7-M1; S1. Using DH5α-pHS-cotG-KERQ7-145E obtained in step (5) as a template, performing one-step full-plasmid PCR site-directed mutagenesis to change the surface amino acid Arg98 to Glu98, thereby obtaining a mutant plasmid pHS-cotG-KERQ7-98E, recorded as pHS-cotG-KERQ7-98E, transforming pHS-cotG-KERQ7-98E into Escherichia coli DH5α, and extracting to obtain the complete plasmid DH5α-pHS-cotG-KERQ7-98E; S2. Using DH5α-pHS-cotG-KERQ7-98E obtained in S1 as a template, a one-step whole plasmid PCR site-directed mutagenesis was performed to change the surface amino acid His36 to Glu36, thereby obtaining a mutant plasmid pHS-cotG-KERQ7-36E, recorded as pHS-cotG-KERQ7-36E. pHS-cotG-KERQ7-36E was transformed into Escherichia coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-36E was obtained by extraction. S3. Using DH5α-pHS-cotG-KERQ7-36E obtained in S2 as a template, a one-step whole plasmid PCR site-directed mutagenesis was performed to change the surface amino acid His17 to Glu17, thereby obtaining a mutant plasmid pHS-cotG-KERQ7-17E, which was recorded as pHS-cotG-KERQ7-M1. The pHS-cotG-KERQ7-M1 was transformed into Escherichia coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-M1 was extracted, wherein the KERQ7 keratinase mutant fragment was recorded as Q7-M1, the nucleotide sequence of Q7-M1 was shown in SEQ ID NO.5, and the amino acid sequence encoded by it was shown in SEQ ID NO.6; S4. Using the amino acid sequence of the keratinase mutant fragment Q7-M1 in S3 as a template, a three-dimensional protein model was constructed, and the obtained protein model was recorded as KERQ7-M1; then, KERQ7-M1 was molecularly docked with the keratin analog AAPF to obtain the KERQ7-M1-AAPF protein ligand complex, and the complex was subjected to molecular dynamics simulation in an acidic pH environment, and the acidic pH environment in the molecular dynamics simulation was 4-6; after simulating the KERQ7-M1-AAPF protein ligand complex in an acidic pH environment, the three-dimensional protein structure model was affected by different environmental pH under different pH environments, and the root mean square fluctuation of amino acids at each site changed differently, and the evaluation of its effect on the acidic environment was based on the change in RMSF; Combined with the RMSF results of molecular dynamics simulation of KERQ7-M1-AAPF in pH environment, the region outside the catalytic pocket of KERQ7-M1 that is most affected by the change in acidic environment is selected, that is, the region with the largest change in RMSF value, which is Glu36~Asn43; (7) Based on the region Glu36 to Asn43 selected in step (6), Glu36 is then used as the insertion site to insert the AAEPVLDTGIE amino acid fragment after the Glu36 site of the keratinase mutant KERQ7-M1 on the pHS-cotG-KERQ7-M1 plasmid to obtain a complete mutant plasmid with the new insertion fragment, which is recorded as pHS-cotG-KERQ7-M2; pHS-cotG-KERQ7-M2 is then transformed into Escherichia coli DH5α, and the DH5α-pHS-cotG-KERQ7-M2 complete plasmid is extracted, wherein the KERQ7 keratinase mutant fragment is recorded as Q7-M2, the nucleotide sequence of Q7-M2 is shown in SEQ ID NO.7, and the amino acid sequence encoded by it is shown in SEQ ID NO.8; (8) transforming the complete plasmid DH5α-pHS-cotG-KERQ7-M2 obtained in step (7) into Bacillus subtilis DB403 to obtain an engineered bacterium B. subtilis DB403 pHS-cotG-KERQ7-M2 that can be used to display keratinase on the surface of spores, inoculating the B. subtilis DB403 pHS-cotG-KERQ7-M2 engineered bacteria into LB medium, and obtaining a culture solution after the first culture; The culture fluid is then inoculated into a DSM liquid culture medium, and after the second culture, the bacterial precipitate is collected by centrifugation, and the precipitate is resuspended using a GTE buffer containing lysozyme, and the resuspended liquid is incubated, and then the precipitate is collected by centrifugation and washed using a PBS buffer, and the collected precipitate after washing is resuspended in a PBS buffer again to obtain a recombinant spore protein suspension with keratinase displayed on the surface; or the precipitate collected after washing is subjected to low-temperature drying to obtain a solid powder; the recombinant spore protein suspension or the solid powder is a keratinase preparation, which is recorded as B. subtilis cotG-KERQ7-M2.

6. The method for preparing an acid-resistant high-activity keratinase preparation according to claim 5, characterized in that: The steps for obtaining cotG-KERQ7 in step (1) are as follows: using the sequence SEQ ID NO.1 of KERQ7 as a template, amplifying KERQ7 with primers Q7-F and Q7-R to obtain an amplified KERQ7 fragment, and then inserting the KERQ7 fragment into the cotG plasmid using two endonucleases, Spe I and Hind III, to construct a recombinant plasmid, which is recorded as cotG-KERQ7; wherein the amplification conditions are: 94°C for 3 min, 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, and 30 cycles; The specific steps for obtaining pHS-cotG-KERQ7-64E in step (3) are as follows: using the shuttle plasmid pHS-cotG-KERQ7 as a template, using primers 64E-F and 64E-R to perform one-step PCR amplification of the pHS-cotG-KERQ7 plasmid, and finally obtaining a mutant plasmid, which is recorded as pHS-cotG-KERQ7-64E; The PCR amplification conditions were as follows: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min for 30 cycles; The sequence of 64E-F is 5'-ccgaatgcgacacaagattttcaatca gag gcacaacatgttgc-3'; The sequence of 64E-R is 5'-gcaacatgttgtgc ctc tgattgaaaatcttgtgtcgcattcgg-3'. In step (4), Alphafold2 is selected for construction of the protein three-dimensional model; the molecular docking of Q7-64E and the keratin analog AAPF is performed by Autodock, and the docking posture is selected to be a conformation in which the catalytic amino acid Ser221 of Q7-64E directly interacts with the AAPF amide bond; wherein the acidic pH environment in the molecular dynamics simulation is 4-6, and the pH environment corresponding to the lowest root mean square deviation is 5; The steps for obtaining pHS-cotG-KERQ7-145E in step (5) are as follows: using plasmid DH5α-pHS-cotG-KERQ7-64E as a template, using primers 145E-F and 145E-R to amplify the pHS-cotG-KERQ7-64E plasmid by one-step PCR, and finally obtaining a mutant plasmid, which is recorded as pHS-cotG-KERQ7-145E; The PCR amplification conditions were as follows: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min for 30 cycles; The sequence of 145E-F is 5'-ttaaaaatgcggtggatacagcgaataat gag ggcgttgttgttgttgttgcag-3'; the primer sequence for 145E-R is 5'-ctgcaacaacaacaacgcc ctc attattcgctgtatccaccgcatt-3'. The step of obtaining pHS-cotG-KERQ7-98E in S1 of step (6) is as follows: using plasmid DH5α-pHS-cotG-KERQ7-145E as a template, using primers 98E-F and 98E-R to amplify pHS-cotG-KERQ7-145E plasmid by one-step PCR, and finally obtaining a mutant plasmid, which is recorded as pHS-cotG-KERQ7-98E; The conditions for PCR amplification were as follows: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min, for 30 cycles; The primer sequence of 98E-F is: 5'-gcctgtatgcggtgaaagttcttgat gag aatggagatggccaat-3'; the primer sequence of 98E-R is: 5'-ccatgaatattggccatctccatt ctc atcaagaactttcaccgcatacag-3'; The step of obtaining pHS-cotG-KERQ7-36E in S2 of step (6) is as follows: using plasmid DH5α-pHS-cotG-KERQ7-98E as a template, using primers 36E-F and 36E-R to amplify pHS-cotG-KERQ7-98E plasmid by one-step PCR, and finally obtaining a mutant plasmid, which is recorded as pHS-cotG-KERQ7-36E; The conditions for PCR amplification were: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min for 30 cycles; The sequence of 36E-F is: 5'-gcatt gag gcagcacatccggatctgaatgttgcagga-3'; The sequence of 36E-R is: 5'-ggatgtgctgc ctc aatgcctgtatccagaactgccac-3'; The steps for obtaining pHS-cotG-KERQ7-M1 in S3 of step (6) are as follows: using plasmid DH5α-pHS-cotG-KERQ7-36E as a template, using primers 17E-F and 17E-R to amplify pHS-cotG-KERQ7-36E plasmid by one-step PCR, and finally obtaining a mutant plasmid, which is recorded as pHS-cotG-KERQ7-M1; The conditions for PCR amplification were: 94°C for 3 min, 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min for 30 cycles; The sequence of 17E-F is 5'-ggcagtg gag gcgcaaggatataaaggagcaaatgt-3'; The sequence of 17E-R is 5'-ccttgcgc ctc cactgccggatccttaatctgttcctg-3'; In step (6), Alphafold2 was selected for constructing the protein three-dimensional model in S4; KERQ7-M1 and the keratin analog AAPF were molecularly docked by Autodock, and the docking posture selected the conformation in which the catalytic amino acid Ser221 of KERQ7-M1 directly interacted with the amide bond of AAPF; the acidic pH environment in the molecular dynamics simulation was 4-6. The concentration of lysozyme in the GTE Buffer in step (8) is 2 mg / mL, wherein the components of the GTE Buffer are: 10 mM EDTA, 20 mM Tris-HCl, pH = 7.5, and 50 mM Glucose; The conditions for the first culture were 37°C, 200 rpm, overnight culture; the conditions for the second culture were 37°C, 200 rpm, 36 h; the conditions for the incubation were 37°C, 60 min, the pH of PBS Buffer was 7.4, and the components of PBS Buffer were (based on 1 L): 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4; the OD of the recombinant spore protein suspension was 600nm is 1-2.

7. An acid-resistant high-activity keratinase preparation prepared according to the method of claim 4 or 5.

8. Use of the acid-resistant high-activity keratinase preparation prepared according to the method of claim 7 for preparing feather meal fermented feed.

9. The use according to claim 8, characterized in that The steps are as follows: the feed raw materials are composed of feather meal, bean dregs and second-rate flour; firstly, fermented seed liquid of brewer's yeast, Bacillus licheniformis, Lactobacillus fermentation and Lactobacillus rhamnosus is inoculated into the feed raw materials, and then an acid-resistant high-activity keratinase preparation B. subtilis cotG-KERQ7-M2, methionine and lysine are added, and finally water is added to the target moisture content to obtain a fermentation system, and coordinated anaerobic fermentation is carried out, and high-quality feather meal fermented feed can be obtained after the fermentation is completed.

10. The use according to claim 8, characterized in that The mass ratio of feather meal, bean dregs and second-grade powder is 4:3:3; the total inoculation amount of fermentation seed liquid of saccharomyces cerevisiae, bacillus licheniformis, lactobacillus fermentation and lactobacillus rhamnosus is 6%, among which the volume ratio of fermentation seed liquid of saccharomyces cerevisiae, bacillus licheniformis, lactobacillus fermentation and lactobacillus rhamnosus is 2:2:1:1; The addition amount of the acid-resistant high-activity keratinase preparation B. subtilis cotG-KERQ7-M2 is as follows: when using spore suspension, the addition amount is 5-15% of the mass of the feed raw material; when using solid powder, the addition amount of the powder is 0.5-1.5% of the mass of the feed raw material; The added amount of methionine is 0.20-0.30% of the mass of the feed raw material, and the added amount of lysine is 0.25% of the mass of the feed raw material; the target water content is 35-45%; the fermentation temperature is 25-40° C., and the fermentation time is 2-14 days.

Images