Preparation method and application of keratinase mutant for efficiently degrading feather feed in acidic gastric juice environment

Abstract

The invention belongs to the field of enzyme engineering and agricultural waste conversion, and particularly relates to a preparation method and application of a keratinase mutant for efficiently degrading feather feed in an acidic gastric juice environment. The method comprises the following steps: replacing amino acid Arg247 with positive charges on the zymoprotein surface based on cotG-KERQ7 in an environment with the pH value of 4, then carrying out rational design on the chemical property of a KERQ7 oxygen negative ion cave, and directly introducing acidic amino acid, so that a mechanism that the serine protease oxygen negative ion cave actively dynamically adjusts the charge density through pH-dependent protonation is introduced, and the pH-dependent protonation of the serine protease oxygen negative ion cave is changed into the pH-dependent protonation of the serine protease oxygen negative ion cave. According to the method, the keratinase is used as a raw material, the oxygen anion hole adaptive low-pH environment mutant is obtained, the acid stability and activity of the keratinase mutant are both improved, an efficient mutant enzyme preparation with activity and acid resistance remarkably improved in the low-pH environment is finally obtained, reference is provided for subsequent improvement of the application value of the keratinase in the feed industry, and the method has a wide application prospect. Wide market application prospects are realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of enzyme engineering and agricultural waste conversion, and particularly relates to a preparation method of a keratinase mutant capable of efficiently degrading feather feed in an acidic gastric juice environment and application thereof. BACKGROUND

[0002] The feather waste generated by the global poultry industry can be as high as tens of millions of tons per year, and its protein content is more than 80%. Improper disposal of the waste not only leads to a huge waste of high-protein resources, but also causes environmental pollution. Although the practice of applying the waste to feed has been carried out for several decades worldwide, the waste is often used as an additive to replace a small amount of fish meal, soybean meal and other nitrogen sources in feed. Although a small amount of feather meal is added, the digestibility of the feather meal is low. Therefore, the feather feed is difficult to promote at present, and the feather protein resource is facing a large amount of waste. The key to limiting the promotion of the feather feed lies in the structure of the feather and the difficulty in being digested by conventional proteases. The feather keratin has a highly cross-linked disulfide bond, hydrogen bond and intermolecular hydrophobic interaction in the molecule. This special structure makes it difficult for traditional proteases such as trypsin, pepsin and papain to efficiently degrade the feather.

[0003]

[0004] Keratinase has a unique mechanism of action. It can not only act on keratin with a tight cross-linked structure, but also directly act on proteins rich in hydrophobic amino acids. Fermentation feed can be prepared by carrying out lactic acid bacteria-keratinase synergistic fermentation on feather meal, so as to improve the palatability, digestibility and utilization rate of the feather meal. In addition, prolonging the retention time of the stomach content can be beneficial to piglets. Improvement of protein hydrolysis in the stomach will lead to a higher protein digestibility in the entire small intestine. However, conventional pepsin cannot efficiently degrade the feather meal. If keratinase has the ability to continuously act in an acidic gastric juice, it can preliminarily digest part of the feather meal in the stomach, so as to drive the further digestion of the feather meal by conventional proteases in the gastrointestinal tract. The above are good directions for utilization of feather resources. However, the enzymatic hydrolysis effect of the feather meal is very poor in the current feed fermentation process, the content of small peptides generated is very low, and there is almost no report on keratinase capable of continuously acting in an acidic gastric juice. The fundamental reason is that keratinase capable of degrading feather has poor acid resistance. At present, not only the mechanism of keratin degradation is not fully elucidated, but also the efficient mechanism of keratinase under acidic conditions is not clear. The research on acid-resistant keratinase is extremely rare internationally. The applicant previously established a three-step rational design based on the enzyme surface for keratinase KERQ7 from Bacillus tequilensis. Although the acid resistance of the KERQ7 mutant is improved, whether the change in electrostatic interaction between the enzyme and the substrate caused by the accumulation of negative charges and the specificity of hydrophobic substrate binding are affected still needs to be explored. Moreover, the obtained mutant cannot completely adapt to the acidic gastric juice environment, and thus needs to be further modified for acid resistance.​

[0005] Oxyanion hole refers to a cavity in the active site of an enzyme that can stabilize the transition state negative charge on deprotonated oxygen or alkoxide, usually composed of a conjugated amide backbone or positively charged residues. In the catalytic process of serine protease, the oxyanion hole acts as a key structural element to stabilize the transition state, stabilizing the negative charge in the reaction intermediate through a hydrogen bond network or electrostatic interaction, thereby reducing the reaction energy barrier and accelerating the catalytic process. In recent years, rational design strategies targeting the oxyanion hole have shown great potential in expanding the substrate spectrum of enzymes, improving catalytic efficiency, and adapting to extreme environments. Zhang et al. studied the PET hydrolytic enzyme by introducing aromatic residues (such as Trp) to enhance the π-π stacking interaction between the oxyanion hole and the rigid polymer substrate, significantly improving its degradation efficiency; Lintuluoto et al. adjusted the topological structure of the oxyanion hole of neurosin by site-directed mutagenesis (S192A), changing the recognition pattern of the polypeptide chain length and charge distribution, thereby improving substrate specificity and catalytic efficiency. Kazuhiro et al. replaced the key residues (such as Ser162) of a hormone-sensitive lipase family of microalkaline archaeal esterase SshEstI with threonine, which has stronger hydrogen bond donor ability, not only reducing the optimal pH (from 8.5 to 7.0), but also improving the thermal stability by optimizing the local hydrophobic interaction. This "single-point multi-function" modification strategy provides a new idea for the design of acid-resistant enzymes. SUMMARY

[0006] To solve the problem that keratinase is difficult to maintain stable and high activity in the acidic environment of lactic acid bacteria feed and the acidic gastric juice of animals in the feather feed industry, the application takes a keratinase KERQ7 mutant Ding-S anchored on the surface coat protein cotG of Bacillus subtilis DB403 as the starting target for further acid-resistant modification. First, the positively charged amino acid Arg247 on the surface of the enzyme protein in the pH = 4 environment is replaced, and then the chemical properties of the oxyanion hole of KERQ7 are rationally designed, and acid amino acids are directly introduced, so that the mechanism of the oxyanion hole of serine protease actively adjusting the charge density through pH-dependent protonation is introduced, and the mutant of the oxyanion hole adapting to low pH environment is obtained, which has outstanding feather meal digestion effect in the stomach of monogastric animals and improves the application value of keratinase in the feed industry.

[0007] To achieve the above application purposes, the technical solutions adopted by the application are as follows:

[0008] (I) A keratinase mutant for efficiently degrading feather feed in acidic gastric juice environment:

[0009] The keratinase mutant KERQ7-123D is formed by mutating arginine (Arg) at position 247 to glutamic acid (Glu) and asparagine (Asn) at position 123 to aspartic acid (Asp) in the keratinase Ding-S mutant sequence shown in SEQ ID NO.2; the nucleotide sequence of the keratinase Ding-S mutant is shown in SEQ ID NO.1.

[0010] The method for preparing the keratinase mutant KERQ7-123D is as follows:

[0011] (1) Using the amino acid sequence SEQ ID NO.2 of the keratinase mutant fragment Ding-S as a template, a three-dimensional protein model was constructed, and the resulting protein model was denoted as Q7-S; subsequently, Q7-S was combined with the keratin analog AAPF (N-succinyl-ala-ala-pro-phe-p-nitroanilide, PubChem CID) Molecular docking was performed using 5496888 to obtain the Q7-S-AAPF protein ligand complex. Molecular dynamics simulations were then conducted on the Q7-S-AAPF protein ligand complex under acidic pH conditions. The root mean square (RMS) fluctuation values ​​of the amino acids at each site in the protein's three-dimensional structure varied under different pH environments. The stability was evaluated by the trend line of the RMS deviation; a lower trend line indicated greater stability. The most stable protein three-dimensional structure model was selected and denoted as M1. Based on the RMS fluctuation values ​​of each amino acid site in model M1, modification sites were selected. The modification site was the surface amino acid Arg247. A mature mutant fragment with Arg247Glu mutation was obtained by modifying the keratinase mutant fragment Ding-S and denoted as KERQ7-N. The nucleotide sequence of KERQ7-N is shown in SEQ ID NO.3, and its encoded amino acid sequence is shown in SEQ ID NO.4.

[0012] (2) Using the amino acid sequence of KERQ7-N in step (1) as a template, a three-dimensional protein model was constructed, and the resulting protein model was denoted as Q7-N. Subsequently, Q7-N was molecularly docked with the keratin analog AAPF to obtain the Q7-N-AAPF protein ligand complex. Molecular dynamics simulations were performed on the Q7-N-AAPF protein ligand complex in an acidic pH environment. The stable three-dimensional structure of the Q7-N-AAPF protein ligand complex after equilibrium was obtained through molecular dynamics simulations, and the resulting stable three-dimensional protein structure model was denoted as M2. Based on model M2, the oxygen-... Using the direct interaction network of Ser125, a key component of the negative ion hole, as a reference, we selected sites that are related to the composition of the oxygen negative ion hole but do not directly interact with it, i.e., secondary interactions. The selected modification site was amino acid Asn123Asp. The resulting mutant was KERQ7-123D, which introduced the negatively charged amino acid Glu into the oxygen negative ion hole, and then introduced an active pH-dependent protonation dynamic regulation mechanism of charge density in the KERQ7-N oxygen negative ion hole. The obtained KERQ7-123D mutant is a keratinase mutant that efficiently degrades feather feed in an acidic gastric juice environment.

[0013] Preferably, in step (1), Alphafold2 is selected for constructing the protein three-dimensional model; Q7-S and keratin analog AAPF are docked through Autodock, and the docking posture is selected as the conformation in which the catalytic amino acid Ser221 of Q7-S interacts directly with the amide bond of AAPF; the acidic pH environment in the molecular dynamics simulation is 4-6, and the pH environment corresponding to the most stable protein three-dimensional structure model is 4.

[0014] Preferably, in step (2), Alphafold2 is selected for constructing the protein three-dimensional model; Q7-N and keratin analog AAPF are docked through Autodock, and the docking posture is selected as the conformation in which the catalytic amino acid Ser221 of Q7-N interacts directly with the amide bond of AAPF; the acidic pH environment in the molecular dynamics simulation is 3-5, and the pH environment corresponding to the stable protein three-dimensional structure model is 3.

[0015] (ii) A mutant gene encoding a keratinase that efficiently degrades feather feed in the acidic gastric juice environment, the sequence of which is as shown in SEQ ID NO.5;

[0016] The method for obtaining the keratinase mutant gene that efficiently degrades feather feed in an acidic gastric juice environment is as follows:

[0017] (1) First, the KERQ7 mutant Ding-S, which is displayed on the surface of the anchoring protein cotG, was selected and denoted as cotG-KERQ7-S. The nucleotide sequence of the mature fragment of Ding-S is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2. Then, cotG-KERQ7-S was integrated into the Escherichia coli-Bacillus subtilis shuttle expression vector pHS to form the shuttle plasmid pHS-cotG-KERQ7-S.

[0018] (2) Using the shuttle plasmid pHS-cotG-KERQ7-S described in step (1) as a template, a one-step whole plasmid PCR site-directed mutagenesis was performed to change the surface amino acid Arg247 to Glu247. The resulting mutant plasmid was named pHS-cotG-KERQ7-N, and the keratinase mutant fragment in the mutant plasmid pHS-cotG-KERQ7-N was named KERQ7-N. The nucleotide sequence of KERQ7-N is shown in SEQ ID NO.3, and the amino acid sequence it encodes is shown in SEQ ID NO.4. Finally, pHS-cotG-KERQ7-N was transformed into Escherichia coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-N was extracted.

[0019] (3) Using the DH5α-pHS-cotG-KERQ7-N obtained in step (2) as a template, one-step whole plasmid PCR site-directed mutagenesis was performed to change the amino acid Asn123, which directly interacts with the key amino acid Ser125 of the oxygen negative ion hole, to Asp123. The resulting mutant plasmid was named pHS-cotG-KERQ7-123D. The keratinase mutant fragment in the mutant plasmid pHS-cotG-KERQ7-123D was named KERQ7-123D. The gene sequence encoding KERQ7-123D was obtained as shown in SEQ ID NO.5.

[0020] Preferably, the steps for obtaining the shuttle plasmid pHS-KERQ7-S in step (1) are as follows: Using the sequence SEQ ID NO.1 of Ding-S as a template, Ding-S is amplified with primers Q7-F and Q7-R to obtain the amplified Ding-S fragment. Then, the Ding-S fragment is inserted into the Escherichia coli-Bacillus subtilis shuttle expression vector pHS-cotG plasmid using SpeI and Hind III restriction enzymes to construct the recombinant shuttle plasmid, denoted as pHS-cotG-KERQ7-S. The amplification conditions are: 94℃ for 3 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, for 30 cycles. The sequence of Q7-F is 5'-ggactagtgctcagacagtgccgtatgg-3'. The sequence of Q7-R is 5'-cccaagcttttaattgctcgccgcctgga-3'.

[0021] Preferably, the specific steps for obtaining pHS-cotG-KERQ7-N in step (2) are as follows: using the shuttle plasmid pHS-cotG-KERQ7-S as a template, the pHS-cotG-KERQ7-S plasmid is amplified by one-step PCR using primers 247E-F and 247E-R, and finally the mutant plasmid is obtained, denoted as pHS-cotG-KERQ7-N;

[0022] The PCR amplification conditions were: 94℃ for 2 min, 98℃ for 10 s, 63℃ for 30 s, 68℃ for 2.5 min, for 30 cycles; the sequence of 247E-F was 5'-cagccaggttcgccagcaactggaaaatacagc-3'; the sequence of 247E-R was 5'-ccagttgctggcgaacctggctgtttgtcag-3'.

[0023] Preferably, the steps for obtaining pHS-cotG-KERQ7-123D in step (3) are as follows: using plasmid DH5α-pHS-cotG-KERQ7-N as a template, the DH5α-pHS-cotG-KERQ7-N plasmid is amplified by one-step PCR using primers 123D-F and 123D-R, and finally the mutant plasmid is obtained, which is denoted as pHS-cotG-KERQ7-123D;

[0024] The PCR amplification conditions were: 94℃ for 3 min, 98℃ for 10 s, 63℃ for 30 s, 68℃ for 2.5 min, for 30 cycles;

[0025] The primer sequence for 123D-F is 5'-atgttattgatatgagcctgggcggaccgaatg-3'; the primer sequence for 123D-R is 5'-cgcccaggctcatatcaataacatccatattatttgcc-3'.

[0026] (III) An engineered bacterium expressing a keratinase mutant that efficiently degrades feather feed in an acidic gastric environment;

[0027] The keratinase mutant engineered bacteria contains the expression vector pHS-cotG-KERQ7-123D; the expression vector pHS-cotG-KERQ7-123D includes a keratinase mutant gene encoding a keratinase mutant that efficiently degrades feather feed in an acidic gastric environment; the keratinase mutant engineered bacteria are obtained by introducing the expression vector pHS-cotG-KERQ7-123D into host cells.

[0028] The steps are as follows: pHS-cotG-KERQ7-123D is transformed into Escherichia coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-123D is extracted; using Bacillus subtilis DB403 as the host bacterium, the recombinant shuttle plasmid DH5α-pHS-cotG-KERQ7-123D is introduced by electroporation to construct the recombinant engineered bacterium B. subtilis DB403 pHS-cotG-KERQ7-123D.

[0029] (iv) Preparation of mutant enzyme preparation B. subtilis DB403 cotG-KERQ7-T2:

[0030] The recombinant engineered bacterium B. subtilis DB403 pHS-cotG-KERQ7-123D was inoculated into a fermentation medium and cultured. After centrifugation, a recombinant spore protein suspension with keratinase on its surface was obtained; or the recombinant spore protein suspension was freeze-dried to obtain a solid powder. The recombinant spore protein suspension or solid powder is a keratinase preparation (i.e., mutant enzyme preparation) that is highly efficient in degrading feather feed in an acidic gastric juice environment, and is designated as B. subtilis DB403 cotG-KERQ7-T2.

[0031] The specific operation is as follows: The engineered bacteria *B. subtilis* DB403 pHS-cotG-KERQ7-123D was inoculated into LB medium and cultured for the first time to obtain a culture solution. The culture solution was then inoculated into DSM liquid medium, and after a second culture, the bacterial precipitate was collected by centrifugation. The precipitate was resuspended using GTE buffer containing lysozyme. The resuspended solution was incubated, centrifuged again, and washed with PBS buffer. The precipitate was then collected again and resuspended in PBS buffer to obtain a recombinant spore protein suspension exhibiting keratinase on its surface. Alternatively, the recombinant spore protein suspension was freeze-dried to obtain a solid powder. Both the recombinant spore protein suspension and the solid powder are keratinase preparations, designated as *B. subtilis* DB403 cotG-KERQ7-T2.

[0032] Preferably, the concentration of lysozyme in the GTE Buffer 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.

[0033] The first culture conditions were 37℃, 200rpm overnight (10-12h); the second culture conditions were 37℃, 200rpm, 36h; the incubation conditions were 37℃, 60min, the pH of the PBS buffer was 7.4, and the composition of the PBS buffer (per 1L) was: 8g NaCl, 0.2g KCl, 1.42g Na2HPO4, 0.27g KH2PO4; the OD600 of the recombinant spore protein suspension was 1-2.

[0034] The keratinase preparation prepared in this invention, which is highly efficient at degrading feather feed in an acidic gastric environment, can be used to further degrade feather meal feed in an acidic gastric environment.

[0035] Simulated gastric fluid (SGF) was prepared according to the United States Pharmacopeia (2000) (2g NaCl, 3.2g pepsin (containing 800-1500 active units per mg), and 7.0mL hydrochloric acid dissolved in 1L, at which point the pH of the solution should be 1.2). 3g of feather meal containing 15-20% keratinase was added to every 100mL of simulated gastric fluid. Because feather meal has a strong acid-binding capacity, it takes a long time for the gastric fluid environment in pigs to return to its normal pH of 1.2 after ingestion. Furthermore, in actual digestion, the pH of animal gastric fluid does not remain consistently at 1.2 most of the time. In the study by Kopper et al., soluble peanut protein was undetectable 1 hour after consuming peanut meal, and the gastric pH only dropped to between 2 and 3 after 0.75 hours. Therefore, in this section, feather meal was added to the prepared simulated gastric fluid to adjust the pH back to 3.5, resulting in a mixture. The mixture was incubated at 37°C and 95 rpm for 1 hour, then dried to constant weight, and the crude protein and small peptide content was determined according to the national standard GB / T 22492-2008.

[0036] The specific steps are as follows:

[0037] (1) Prepare artificial gastric juice according to the United States Pharmacopeia: 2g NaCl, 3.2g pepsin (each mg of pepsin contains 800-1500 active units), 7mL hydrochloric acid, dissolved in deionized water to make up to 1L, the pH of the solution is 1.2;

[0038] (2) Add keratinase preparation and feather powder to artificial gastric juice and adjust the pH to obtain a mixture. After incubation, the mixture can be used to efficiently degrade feather feed in an acidic gastric juice environment.

[0039] Preferably, in step (2), the ratio of feather powder to artificial gastric fluid is 3g:100mL; when the keratinase preparation is a recombinant spore protein suspension, the amount added is 15%-20% of the total amount of feather powder, i.e., 15-20ml:100g; when the keratinase preparation is a solid powder, the amount added is 15%-20% of the total amount of feather powder, i.e., 15-20g:100g; the pH is adjusted to 3.5; the incubation conditions are: 37℃, 95rpm for 1h.

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

[0041] (1) This invention establishes a rational design concept for acid resistance based on the oxygen anion hole of keratinase. This invention is one of the very few studies in the world that modifies the acid resistance of keratinase and is original. In this study, the positively charged amino acid Arg247 on the surface of the spore keratinase cotG-KERQ7 in a pH=4 environment was replaced. Then, the chemical properties of the oxygen anion hole of KERQ7 were rationally designed, and acidic amino acids were directly introduced. This introduced the mechanism by which the oxygen anion hole of serine protease actively regulates the charge density through pH-dependent protonation. This mechanism enhances the stability and activity of KERQ7 in acidic gastric juice, and finally, the keratinase mutant T2 was obtained. T2 has better low pH acid stability and activity, as well as better ability to degrade feather meal in acidic gastric juice, providing a technical solution for the development of acidic keratinase and its further application in the feed industry.

[0042] (2) This invention utilizes the above-mentioned rational design method based on the oxygen anion hole of keratinase to achieve rational modification of keratinase to tolerate the gastric acid environment, and finally obtains a highly efficient spore-forming enzyme preparation with significantly improved activity and acid resistance under low pH conditions. Furthermore, the spore-forming enzyme preparation prepared by this invention can be directly added in the form of spore suspension, or it can be dried at low temperature to make it into a solid powder, making it easier to store and transport. Attached Figure Description

[0043] Figure 1 This is a bioinformatics analysis of the enzyme activity of the mutant Ding-N; (a) shows a comparison of enzyme activities of the Ding-S and Ding-N mutant spore enzyme preparations; (b) shows the residual activity of the Ding-N spore enzyme preparation after incubation for 1 and 3 hours at different pH environments; (c) shows the interaction network before and after the mutation at site 247, where the blue dashed line represents hydrogen bonds and the gray dashed line represents van der Waals forces; (d) shows the properties of the S1 substrate binding pocket of Ding-N and Ding-S; (e) shows a comparison of the RMSD of the backbone of Ding-N after MD simulation at different pH environments; (f) shows a comparison of the RMSF of the residues of Ding-N after MD simulation at different pH environments.

[0044] Figure 2 It is the amino acid network directly affected by Ser125 (left image) and Ser224 (right image), which are key components of the oxygen negative ion hole.

[0045] Figure 3 Figure (a) shows a comparison of enzyme activities of spore enzyme preparations from B. subtilis DB403 pHS-cotG-KERQ7-N (labeled Ding-N) and B. subtilis DB403 pHS-cotG-KERQ7-T2 (labeled T2).

[0046] (b) The figure shows the residual activity of the B. subtilis DB403 pHS-cotG-KERQ7-T2 mutant spore enzyme preparation after incubation for 1 and 3 hours at different pH values.

[0047] Figure 4 This is a comparison of the RMSD of the T2-AAPF complex skeleton after MD simulation in different pH environments.

[0048] Figure 5 This is a diagram showing the dynamic changes in hydrogen bonds generated by Glu64 in mutants T2 and Ding-N at pH=3.

[0049] Figure 6 This is a dynamic diagram of the hydrogen bond changes between solute and solvent in the T2-AAPF complex under different pH environments, simulated by MD.

[0050] Figure 7 This is a comparison of the enzyme activities of spore enzyme preparations from the mutants B. subtilis DB403 pHS-cotG-KERQ7-T2 (marked T2 in the figure) and B. subtilis DB403 pHS-cotG-KERQ7-A152V (marked A152V in the figure).

[0051] Figure 8 The structure of the A152V-AAPF complex was observed after a 50 ns MD simulation at pH 3.

[0052] Figure 9 The comparison of RMSF of B. subtilis DB403 pHS-cotG-KERQ7-T2 (marked T2 in the figure) and B. subtilis DB403 pHS-cotG-KERQ7-A152V (marked A152V in the figure) after 50 ns MD simulation in an environment of pH=3; the blue box shows the RMSF of catalytic Ser221.

[0053] Figure 10 The effect of enzyme preparations on the content of small peptides produced by digesting feather meal in an artificial gastric juice environment. Figure 10 Different lowercase letters indicate significant differences (P<0.05).

[0054] Note: In the above figure, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001, and ns indicates no significant difference. Detailed Implementation

[0055] The invention is further defined in the following embodiments. Based on the above description and these embodiments, those skilled in the art can determine the basic features of the invention, and various modifications can be made to the invention without departing from the spirit and scope of the invention to make it suitable for various uses and conditions.

[0056] The Escherichia coli-Bacillus subtilis shuttle expression vector pHS, Escherichia coli DH5α, Escherichia coli BL21(DE3), and Bacillus subtilis DB403 and cotG-KERQ7 are all well-known in the field, and their acquisition methods can be found in patent CN119685295A.

[0057] DSM liquid medium (Difco Sporulation Medium) consists of: 0.8% Difco broth, 0.1% KCl, 0.025% MgSO4·7H2O, 1.0 mM Ca(NO3)2·4H2O, 10 μM / L MnCl2, and 1.0 μM / L FeSO4.

[0058] In the evaluation of the degradation effect of feather meal in the gastric acid environment, the determination methods for crude protein and small peptide content refer to the national standard GB / T 22492-2008.

[0059] The following embodiments relate to a method for determining the activity of keratinase using feather meal as a substrate, specifically as follows: Take 1.0 mL of spore enzyme preparation (spore suspension, adjusted OD...) 600nm =1.0), add 2.0 mL of the appropriate pH buffer (disodium hydrogen citrate-citrate buffer (phosphate buffer) for pH 3-7); then add 10 mg of feather meal as substrate, react in a shaker at 30℃ or 40℃, add 2.0 mL of 10% trichloroacetic acid (TCA) after 1 h to terminate the reaction, centrifuge at 10000 rpm at 4℃ for 10 min, and determine the OD value of the supernatant. 280nm The absorbance at 280 nm was measured, and reaction tubes treated with TCA before the reaction were used as controls. Enzyme activity was defined as a 0.01 increase in absorbance at 280 nm under the above reaction conditions, which was defined as one unit of enzyme activity (U).

[0060] Implementation Instructions:

[0061] (1) The molecular dynamics simulations involved in this invention all adopted the following steps: the protonation states of Glu and Asp in the protein structure were predicted using the PDB 2QR online server (https: / / server.poisonboltzmann.org / pdb2pqr), and written to the file to allocate charges. Molecular dynamics (MD) simulations of the protonated protein-ligand complex were performed using GROMACS (version 2024.4), employing the TIP 3P water model and AMBER 14 all-atom force field. The protein-ligand complex was placed in a 263 nm volume... 3 In the dodecahedral box (the distance from the box boundary to the surface of the composite is...) Then fill with TIP 3P water molecules. Add sodium ions (Na+). + The system contains chloride ions (Cl-) to maintain electroneutrality and equilibrium. Next, a 5000-step energy minimization process (0.002 ps per step) is performed at 298.15 K, using a steepest descent algorithm to optimize the system and remove unreasonable geometries. Then, a 2 ns equilibrium simulation is performed at 298.15 K and 1 bar, with positional constraints imposed on the protein to allow for sufficient relaxation of water molecules. After the system reaches equilibrium, the positional constraints are removed, and a 50 ns dynamics simulation is performed. Periodic boundary conditions are applied to the trajectory data, and the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) are analyzed. The binding free energy of the complex is estimated using a method combining molecular mechanics calculations and Poisson-Boltzmann electrostatics (MM-PBSA). The gmx_mmpbsa tool is used to extract stable trajectory data from the molecular dynamics simulation for MM-PBSA calculations.

[0062] (2) The primer sequences involved in the implementation of this invention are shown in the table below:

[0063] Table 1 Primer sequences

[0064] Primer name Primer sequence P1-F 5'-gttggtagctcttgatccgg-3' P1-R 5'-gttgctgttcctgttctgaat-3' 247E-F 5'-cagccaggttcgccagcaactggaaaatacagc-3' 247E-R 5'-ccagttgctggcgaacctggctgtttgtcag-3' 123D-F 5'-atgttattgatatgagcctgggcggaccgaatg-3' 123D-R 5'-cgcccaggctcatatcaataacatccatattatttgcc-3' 152V-F 5'-gttgttgttgttgcagtagcgggaaatagcggc-3' 152V-R 5'-atttcccgctactgcaacaacaacaacgcc-3'

[0065] Example 1: Further stabilization of the enzyme protein surface of keratinase KERQ7 in a low pH environment

[0066] (1) In this invention, the KERQ7 mutant Ding-S, which is displayed on the surface of the anchoring protein cotG, is selected and designated as cotG-KERQ7-S. The nucleotide sequence of the mature fragment of Ding-S is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2. The cotG-KERQ7-S is integrated into the Escherichia coli-Bacillus subtilis shuttle expression vector pHS to form the shuttle plasmid pHS-cotG-KERQ7-S. The shuttle plasmid pHS-cotG-KERQ7-S is transformed into Escherichia coli DH5α and DH5α-pHS-cotG-KERQ7-S is extracted.

[0067] The steps for obtaining pHS-KERQ7-S are as follows: Using the sequence SEQ ID NO.1 of Ding-S as a template, Ding-S was amplified with primers Q7-F and Q7-R (conditions: 94℃ for 3 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles); then, Ding-S was inserted into the Escherichia coli-Bacillus subtilis shuttle expression vector pHS-cotG plasmid using Spe I and Hind III restriction enzymes to construct the recombinant shuttle plasmid, denoted as pHS-cotG-KERQ7-S; Q7-F: 5'-ggactagtgctcagacagtgccgtatgg-3'; Q7-R: 5'-cccaagcttttaattgctcgccgcctgga-3'.

[0068] (2) Using the amino acid sequence of the keratinase mutant fragment Ding-S as a template, Alphafold2 was selected to construct a three-dimensional protein model, and the resulting protein model was denoted as Q7-S. Subsequently, Q7-S was molecularly docked with the keratin analog AAPF (N-succinyl-ala-ala-pro-phe-p-nitroanilide, PubChem CID 5496888) to obtain the Q7-S-AAPF protein ligand complex.

[0069] Molecular dynamics simulations were performed on the Q7-S-AAPF protein-ligand complex under acidic pH conditions (pH = 4-6). The root mean square fluctuation (RMSF) values ​​of amino acids at different points in the protein's three-dimensional structure varied under different pH conditions. Stability was evaluated by the trend line of the RMSF, with lower values ​​indicating greater stability. Simulation results showed that the protein was most stable at pH = 4; therefore, the protein three-dimensional structure model at pH = 4 was selected and denoted as M1. The trajectory files obtained from the molecular dynamics simulations of Q7-S-AAPF, including RMSD and RMSF values, were analyzed using VMD 1.9.3. The static structure and function of Q7-S-AAPF were visualized using Pymol 2.5.5. The gmx_mmpbsa tool was used to extract stable trajectory data from the molecular dynamics simulations for MM-PBSA calculations.

[0070] (3) Further, based on the root mean square fluctuation (RMSF) value of each amino acid site in the protein three-dimensional structure model M1, the modification site is selected; the amino acids that are unstable (large RMSF variation) and positively charged on the surface of the enzyme protein are replaced; the modification site is the surface amino acid Arg247.

[0071] (4) Using the shuttle plasmid DH5α-pHS-cotG-KERQ7-S in step (1) as a template, the pHS-cotG-KERQ7-S plasmid was amplified by one-step PCR using primers 247E-F and 247E-R, and finally the mutant plasmid was obtained, which was denoted as pHS-cotG-KERQ7-N.

[0072] The PCR amplification conditions were: 94℃ for 2 min, 98℃ for 10 s, 63℃ for 30 s, 68℃ for 2.5 min, for 30 cycles; the sequence of 247E-F was 5'-cagccaggtt. cgc cagcaactggaaaatacagc-3'; the sequence of 247E-R is 5'-ccagttgctg gcg aacctggctgtttgtcag-3'.

[0073] The KERQ7-N keratinase mutant fragment in the mutant plasmid pHS-cotG-KERQ7-N is designated as KERQ7-N; the nucleotide sequence of KERQ7-N is shown in SEQ ID NO.3, and the amino acid sequence it encodes is shown in SEQ ID NO.4; finally, pHS-cotG-KERQ7-N is transformed into Escherichia coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-N is extracted;

[0074] (5) Using the amino acid sequence of the keratinase mutant fragment KERQ7-N in step (4) as a template, Alphafold2 was selected to construct a three-dimensional protein model, and the resulting protein model was denoted as Q7-N. Subsequently, Q7-N was molecularly docked with the keratin analog AAPF (N-succinyl-ala-ala-pro-phe-p-nitroanilide, PubChem CID 5496888) to obtain the Q7-N-AAPF protein ligand complex. Molecular dynamics simulations were performed on this complex in an acidic pH environment (pH=3-5). The simulation results showed that it was most stable at pH=3. Therefore, the three-dimensional protein structure model at pH=3 was selected and denoted as M2.

[0075] The trajectory files obtained from molecular dynamics simulations of Q7-N-AAPF, including root mean square deviation (RMSD) and root mean square fluctuation (RMSF), were analyzed using VMD 1.9.3. The static structure and function of Q7-N-AAPF were visualized using Pymol 2.5.5. The gmx_mmpbsa tool was used to extract stationary trajectory data from the molecular dynamics simulations for MM-PBSA calculations.

[0076] This example compares the optimal pH of the KERQ7 mutant Ding-S, which displays the capsid protein cotG on the surface of Bacillus subtilis spores, with the surface-stabilized mutant KERQ7-N using dotted amino acids. The complete plasmids DH5α-pHS-cotG-KERQ7-S and DH5α-pHS-cotG-KERQ7-N were then transformed into Bacillus subtilis DB403 to obtain engineered bacteria B. subtilis DB403 pHS-cotG-KERQ7-S and B. subtilis DB403 pHS-cotG-KERQ7-N, which can be used to display keratinase on the spore surface.

[0077] Preparation of keratinase enzyme preparation: *B. subtilis* DB403 pHS-cotG-KERQ7-S and *B. subtilis* DB403 pHS-cotG-KERQ7-N were inoculated into LB medium at a 1% inoculum and cultured overnight (12 h) at 37°C and 200 rpm to obtain the culture medium. The culture medium was then inoculated into DSM liquid medium at a 1% inoculum and cultured at 37°C and 200 rpm for 36 h. The bacterial cell pellet was collected by centrifugation, and the pellet was resuspended in GTE buffer containing 2 mg / mL lysozyme. The resuspended pellet was incubated, centrifuged, and washed with PBS buffer. The pellet was then resuspended in PBS buffer again to obtain a recombinant spore protein suspension (OD) displaying keratinase on its surface. 600nm 1), that is, the keratinase enzyme preparations are obtained, which are respectively denoted as B. subtilis cotG-KERQ7-S (abbreviated as Ding-S) and B. subtilis cotG-KERQ7-N (abbreviated as Ding-N);

[0078] The GTE Buffer consists of 10 mM EDTA, 20 mM Tris-HCl (pH 7.5), and 50 mM Glucose. The PBS Buffer has a pH of 7.4 and consists of (per 1 L) 8 g NaCl, 0.2 g KCl, 1.42 g Na₂HPO₄, and 0.27 g KH₂PO₄.

[0079] The keratinase enzyme preparations of B. subtilis DB403 cotG-KERQ7-S and B. subtilis DB403 cotG-KERQ7-N were tested and compared at pH 4, 5, and 6. The procedure was as follows:

[0080] S1. The spore count of the two keratinase enzyme preparations was adjusted to 1×10⁻⁶ using a serial dilution coating plate method. 8 cells / mL;

[0081] S2. Then, the adjusted B. subtilis DB403 cotG-KERQ7-S keratinase preparation from step S1 was added to phosphate buffer, followed by feather meal as a substrate. After incubation at a constant temperature, trichloroacetic acid (TCA) was added to terminate the reaction. After centrifugation, the supernatant was collected to determine the OD. 280nm The absorbance at 280 nm was measured; the control group was treated with TCA before the isothermal reaction, and the other operations were the same; 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 unit of enzyme activity (U).

[0082] In this step, the ratio of B. subtilis DB403 cotG-KERQ7-S keratinase enzyme preparation, phosphate buffer, feather meal, 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 reaction was carried out at 37°C in a shaker for 1 h; and the centrifugation was carried out at 4°C and 10,000 rpm for 10 min.

[0083] The phosphate buffer solutions have a pH range of 4-6. The pH 4 solution consists of 7.71 mL of 0.2 M Na₂HPO₄ and 12.29 mL of 0.1 M citric acid solution; the pH 5 solution consists of 10.3 mL of 0.2 M Na₂HPO₄ and 9.7 mL of 0.1 M citric acid solution; and the pH 6 solution consists of 12.63 mL of 0.2 M Na₂HPO₄ and 7.37 mL of 0.1 M citric acid solution.

[0084] S3 is the same as step S2, except that B. subtilis DB403 cotG-KERQ7-S keratinase preparation is replaced with B. subtilis DB403 cotG-KERQ7-N keratinase preparation.

[0085] from Figure 1 As shown in Figure (a), the optimal pH for the mutant (Ding-N) shifted to around 4, and its activity at pH 4 was equivalent to 158.3 ± 1.55% of that of Ding-S. After incubation for 3 hours at pH 3.5, pH 4, and pH 4.5, it still retained 83.36 ± 4.912%, 86.65 ± 1.75%, and 86.02 ± 1.798% of its activity, respectively. Figure 1 (b) The experimental results show that Ding-N has further improved acid resistance and activity. The following section provides bioinformatics verification and analysis. First, the three-dimensional structure of the mutant Ding-N was predicted and evaluated. The insertion site of the substrate AAPF was determined using Autodock molecular docking. Then, molecular dynamics simulations of the KERQ7-AAPF complex system were performed under different pH conditions (pH = 3, 4, 5). The equilibrium structure after simulation was demonstrated and observed using Pymol. The RMSF results of Ding-S and Ding-N were derived after the simulation, showing a significant decrease in instability at this site (from...). Descending to Subsequently, the binding energy change ΔΔG resulting from the Glu247→Arg point mutation was predicted using the DUET online tool to be -0.213 kcal / mol. This indicates that glutamate at position 247 has a higher conformational entropy than arginine, making this site more stable. Combined with the enzyme protein structure obtained from molecular dynamics simulations, new hydrogen bonds are formed between Glu247 and Asn243 and Glu251, more than the number of hydrogen bonds at this site in Ding-S. Furthermore, the van der Waals forces between Glu247 and Leu250 and Glu251, along with the hydrogen bonds, synergistically stabilize the α-helical region where they reside. Therefore, Ding-N exhibits improved acid resistance compared to Ding-S. Figure 1 (c) For protein structure, large and deep hydrophobic cavities are crucial for ligand binding. Therefore, the protein equilibrium structures of both proteins after 50 ns of simulation in a pH=4 environment were derived, and the S1 substrate binding pocket portion of both proteins was calculated using the Protein-plus online server. This pocket, which is the location of the catalytic dual-component (not the entire substrate binding pocket), showed that the S1 substrate binding pocket of Ding-N is deeper than that of Ding-S. And it has a larger pocket surface area and larger pocket capacity This results in increased viability and stability in a pH=4 environment. Figure 1 (d) However, the activity and acid stability of the mutant Ding-N decreased sharply at pH 3.0. The following attempts to analyze the reaction mechanism of this phenomenon using molecular dynamics simulation results. MD simulation results of Ding-N under different pH conditions show that, after the complex stabilizes, the average RMSD at pH 3 is lower than the RMSD at pH 4 and pH 5. Figure 1 (e) indicates that the AAPF-Ding-N complex has stronger stability in an environment with pH=3. However, the RMSF of 64Glu in the catalytic diastereomer is 0.0771 nm, 0.1374 nm, and 0.0982 nm, respectively, when the ambient pH is 3, 4, and 5. Figure 1 (f)); Then, MM / PBSA analysis was performed on the AAPF-Ding-N complex system after simulation stabilization. The results showed that the electrostatic interaction between AAPF and Ding-N (ΔE) was significant at pH = 3. ele The concentration of AAPF at pH = 675.35 kcal / mol indicates a strong attraction between the substrate and the ligand, while the electrostatic interaction between AAPF and Ding-N at pH = 4 (ΔE) is also significant. ele The concentration was -339.12 kcal / mol. This was combined with RMSF results, MM / PBSA analysis results, and the apparent pK of glutamate. aA value of 4.25 indicates that the AAPF-Ding-N complex at pH 3 exhibits relatively stronger stability—more accurately, stronger rigidity. This may be due to the excessive protonation of 64Glu in the catalytic diamagnetic, which enhances the electrostatic attraction between the ligand and the positively charged residues, leading to conformational rigidity and thus being less conducive to catalysis. At pH 4, however, the protonation state reaches a relative equilibrium, exhibiting both a relatively moderate electrostatic attraction (ΔE) and... ele The absolute value is significantly lower than that at pH 3, while maintaining conformational flexibility. However, although the activity of Ding-N is enhanced at pH 4, the MD simulation results at pH 4 show a larger ΔE. ele Absolute values ​​can also lead to excessive desolvation, resulting in binding instability. The root cause is that KERQ7 is not yet well adapted to this low pH environment, requiring optimization of the charge distribution or protonation state of the protein ligand binding site. Considering this phenomenon, and taking into account that under low pH conditions, the increased protonation of the enzyme's active site leads to a decrease in transition state stability, further improving the low pH stability and activity of KERQ7 should start with the oxygen anion hole, neutralizing local charges, improving the protein ligand binding instability caused by excessive desolvation, and enhancing the interaction with oxygen anions. Therefore, the next step will be to rationally modify the oxygen anion hole charge of Ding-N.

[0086] In summary, the results of Example 1 show that, considering the increased protonation of enzyme active sites under low pH conditions, which leads to a decrease in transition state stability, if we want to further improve the low pH stability and activity of KERQ7, we should start with the oxygen anion hole, neutralize the local charge, reduce dependence on environmental pH, and enhance the interaction with oxygen anions. Therefore, the next step will be to rationally modify the oxygen anion hole charge of Ding-N.

[0087] Example 2: Effect of Ding-N oxygen anion hole introducing negative charge on acid resistance

[0088] This example demonstrates how surface amino acids of the mutant Ding-N from Example 1 were modified to obtain the mutant KERQ7-123D.

[0089] (1) The structure of the protein three-dimensional structure model M2 in step (5) of Example 1 was observed. The amino acid Asn123 in the direct interaction network with the key amino acid Ser125 of the oxygen negative ion hole was changed to Asp123, and the mutant plasmid was obtained as pHS-cotG-KERQ7-123D. The KERQ7-123D keratinase mutant fragment in the mutant plasmid pHS-cotG-KERQ7-123D was named KERQ7-123D. The nucleotide sequence of KERQ7-123D is shown in SEQ ID NO.5, and the amino acid sequence it encodes is shown in SEQ ID NO.6. Then, pHS-cotG-KERQ7-123D was transformed into Escherichia coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-123D was extracted.

[0090] The PCR amplification conditions were: 94℃ for 3 min, 98℃ for 10 s, 63℃ for 30 s, 68℃ for 2.5 min, for 30 cycles;

[0091] The primer sequence used for 123D-F is 5'-atgttattgatatgagcctgggcggaccgaatg-3'; the primer sequence for 123D-R is 5'-cgcccaggctcatatcaataacatccatattatttgcc-3'.

[0092] (2) Using the amino acid sequence of the keratinase mutant fragment KERQ7-123D in step (1) as a template, Alphafold2 was selected to construct a three-dimensional protein model, and the resulting protein model was denoted as Q7-123D. Subsequently, Q7-123D was molecularly docked with the keratin analog AAPF (N-succinyl-ala-ala-pro-phe-p-nitroanilide, PubChem CID5496888) to obtain the Q7-123D-AAPF protein ligand complex. Molecular dynamics simulations were performed on this complex in an acidic pH environment (pH=3-5). The results showed that it was most stable at pH=3. Therefore, the three-dimensional protein structure model at pH=3 was selected and denoted as M3. The trajectory files obtained from molecular dynamics simulations of Q7-123D-AAPF, including root mean square deviation (RMSD) and root mean square fluctuation (RMSF), were analyzed using VMD 1.9.3. The static structure and function of Q7-N-AAPF were visualized using Pymol 2.5.5. The gmx_mmpbsa tool was used to extract stationary trajectory data from the molecular dynamics simulations for MM-PBSA calculations.

[0093] This example describes the mutation process from KERQ7-N to KERQ7-123D, and compares the acid stability and activity of the keratinase mutant B. subtilis DB403 cotG-KERQ7-N with further stabilized surface charge in Example 1 with the keratinase mutant B. subtilis DB403 cotG-KERQ7-123D with negative charge introduced by oxygen anion holes.

[0094] The spore enzyme preparation B. subtilisDB403 cotG-KERQ7-123D was prepared according to the method for preparing keratinase enzyme preparation in step (5) of Example 1, except that the complete plasmid DH5α-pHS-cotG-KERQ7-N was replaced with DH5α-pHS-cotG-KERQ7-123D to construct the recombinant engineered bacterium B. subtilisDB403 pHS-cotG-KERQ7-123D; finally, the keratinase enzyme preparation was obtained, which was denoted as B. subtilisDB403 cotG-KERQ7-T2;

[0095] Using the same method for determining the degradation activity of feather meal as described in Example 1, the changes in the degradation activity of feather meal by B. subtilis DB403 cotG-KERQ7-N (abbreviated as Ding-N in the figure) and B. subtilis DB403 cotG-KERQ7-T2 (abbreviated as T2 in the figure) in different pH (3-5) environments were compared.

[0096] The choice of directly introducing a negative charge into the oxyanion hole of serine proteases aims to neutralize the negative charge in the transition state under acidic conditions, thus making it more stable in acidic environments. In the catalytic mechanism of serine proteases, the oxyanion hole is usually formed by several skeletal amide groups. In Ding-N, the key amino acids corresponding to the formation of the oxyanion hole are Ser221, Ser125, and Ser224. Among them, Ser221 and Ser125 form hydrogen bonds to stabilize the oxyanion in the transition state. Introducing a negatively charged amino acid into this region may generate electrostatic repulsion, which is detrimental to stabilizing the negatively charged transition state. Structural observation after equilibrium observation of Ding-N in a pH=3 environment using molecular dynamics simulation revealed van der Waals forces between Ser224 and Ser125, which forms the oxyanion hole. This non-bonded interaction indicates that Ser224 contributes to the stability of the oxyanion hole, while a hydrogen bond is formed between Ser224 and the amide bond of the Asn123 side chain. Figure 2 This indicates that Asn123 is located near the oxygen anion hole and does not directly participate in the catalytic triplet. Therefore, we performed a site-directed mutagenesis on Asp to replace the neutral Asn and thus neutralize the oxygen anion.

[0097] Subsequently, the enzyme activity and acid tolerance of Asn123→Asp(T2) were measured; the results are as follows: Figure 3 As shown, the KERQ7-123D mutant exhibited increased activity in acidic environments, with its activity at pH 3 equivalent to 180.36 ± 1.156% of that of Ding-N. Furthermore, after 3 hours of incubation at pH 3, it still retained 92.308 ± 4.912% of its activity. KERQ7-123D also showed good tolerance to incubation at other acidic gradients. Figure 3 (b) This experimental result demonstrates that the mutant KERQ7-123D has further improved acid resistance and activity. The following is a bioinformatics analysis and verification of this effect. The transformation from Ding-N to the KERQ7-123D mutant mainly enhances activity and stability at pH=3. This phenomenon is analyzed and discussed below. First, molecular dynamics simulations of the KERQ7-123D-AAPF complex were performed at pH=3, 4, and 5 for 50 ns. The side chain of asparagine is an amide group (-CONH2), while aspartic acid is a carboxylic acid group (-COOH). The apparent pK a The value is around 3.9. In an environment with pH=3, the aspartic acid side chain partially protonates, reducing the charge of the carboxylate group and eliminating the repulsion caused by electrostatic effects. This allows the enzyme to maintain its structure and function in an acidic environment. Furthermore, the mutated aspartic acid acts as a proton donor during catalysis in an acidic, low-pH environment, helping to neutralize the negative charge in the transition state and promoting its stability, thereby enhancing the catalytic effect. Based on the MM / PBSA analysis results of the MD simulation process of KERQ7-123D-AAPF in various acidic gradients (pH=3 / 4 / 5) after equilibrium, it was found that the ΔG of KERQ7-123D-AAPF at pH4 is... tot The most negative value, reaching -36.68, theoretically indicates the most stable binding, consistent with the experimental results showing the highest activity. The ΔG values ​​at pH=3 and pH=5 are also relevant. tot The values ​​are quite close, at -34.52 and -34.13 respectively. However, the activity enhancement of the KERQ7-123D mutant at pH 3 is significant. Therefore, to analyze the mechanism of the increased catalytic efficiency of the KERQ7-123D mutant at pH 3, in addition to considering binding capacity, other factors need to be analyzed. From the perspective of hydrophobic interactions, the ΔE of the KERQ7-123D mutant at pH 3... vdW The -63.51 energy level is significantly stronger than Ding-N's -18.53, indicating that KERQ7-123D exhibits more stable hydrophobic interactions under acidic conditions at pH 3, potentially enhancing ligand binding. This is further supported by the nonpolar solvation energy (ΔE). np From this perspective, the ΔE of the KERQ7-123D npA more negative contribution (-5.56 < -3.00) in a pH 3 environment indicates a more compact hydrophobic core. ΔE vdW and ΔE np The superior performance under acidic conditions suggests that the enhanced hydrophobic core and van der Waals contacts may maintain protein folding stability, improving the pH 3 acid stability and activity of the KERQ7-123D mutant. MM / PBSA analysis showed that Ding-N exhibited better ΔE at pH 3. ele The abnormally negative value (-675.35) may be due to excessive desolvation leading to unstable binding. This phenomenon was also observed in simulations of Ding-N at pH=4 (ΔE). ele =-339.12), the fundamental reason is that the protonated state of Ding-N at lower pH (3-4) is not suitable for the corresponding environment. After introducing the aspartic acid mutation, the electrostatic interaction ΔE between the substrate AAPF and the mutant KERQ7-123D in a pH 4 environment is reduced. ele ΔE is -47.67 in an environment with pH=3. ele The value is -47.54, combined with the simulation results, the polar solvation energy ΔG PB Let's look at ΔG PB At pH 3, the value was 82.35, significantly lower than Ding-N's 667.18, ΔG PB At pH 4, the value of 78.34 was significantly lower than that of Ding-N (346.37), indicating that its binding process is less dependent on solvation. This may be achieved by reducing the exposure of polar residues. Therefore, KERQ7-123D optimizes the contradiction of excessive desolvation by adjusting the charge distribution or protonation state, thus improving its stability at low pH. The binding and catalytic advantages of the mutant KERQ7-123D under acidic conditions were analyzed based on MM / PBSA results, and its ΔG... tot The levels at pH 3 (-34.25) and pH 4 (-36.68) were significantly lower than those of the wild type (pH 3: -29.70; pH 4: -11.56), indicating that the mutant optimized electrostatic interactions (ΔE). ele (Tending towards normal) and van der Waals force (ΔE) vdW (Stronger) enhanced substrate binding ability, consistent with experimental results showing increased acid tolerance and enhanced activity. Of particular note is that Ding-N, despite ΔG at pH=4, showed improved substrate binding ability. tot The binding strength was relatively high (weak), but the experimental activity reached its peak, suggesting that its catalytic efficiency is not solely dependent on binding strength, but may also be related to the protonation state or substrate binding mode. Furthermore, the protonation state of the T2 mutant after introducing an aspartic acid mutation has a positive impact on the overall structure and charge (ΔE). ele From -339.12 to -47.67, ΔG PB(From 346.37 to 78.34), further indicating that the mutant KERQ7-123D reduces the polar solvation energy (ΔE). np This reduces dependence on solvation while balancing charge distribution at low pH levels (alleviating the ΔE of Ding-N at pH 3-4). ele (abnormally negative values), thereby stabilizing the enzyme-substrate complex, improving catalytic efficiency in acidic environments, and enhancing acid stability. (Binding) Figure 1 (e) Figure 4 and Figure 5 The number of hydrogen bonds generated by the catalytic residue Glu64 after the protein-ligand complex stabilized in a pH=3 environment was observed. The number of Glu64 hydrogen bonds in KERQ7-123D after structural stabilization was significantly lower than that in Ding-N, indicating that the excessive protonation of the catalytic residue was alleviated after the introduction of acidic amino acids into the oxygen anion hole. Next, the dynamic changes in hydrogen bonds between the solute and solvent were observed during the simulation. The changes in hydrogen bonds between the solute and solvent in the KERQ7-123D mutant after MD simulation at various pH gradients were observed (see graph). Figure 6 It was observed that the KERQ7-123D-AAPF complex exhibits a significantly higher number of hydrogen bonds at pH 4. Hydrogen bonds are typically associated with protein stability, and the increased number of hydrogen bonds between the solvent and solute likely stabilizes the enzyme's surface conformation, reducing structural fluctuations caused by pH changes. This stability is crucial for maintaining the precise geometric arrangement of active sites. These findings also indicate that the KERQ7-123D mutant protein-ligand complex has higher solubility and better adaptability to this low pH environment compared to the Ding-N complex.

[0098] The isoelectric point of KERQ7-123D was predicted to be 4.012 using Novopro's online tool (https: / / www.novopro.cn / tools / protein_iep.html). Combined with simulation results, it can be concluded that pH=4 is close to the isoelectric point of KERQ7-123D. At this pH, the change in charge distribution may promote the formation of more short-range ion pairs, regulate the proton transport pathway, stabilize the transition state intermediate, and the hydrogen bond network may form a solvent shell around the active site, optimizing the local microenvironment and thus improving catalytic efficiency.

[0099] Conclusion: The results of Example 2 show that the introduction of a negatively charged aspartic acid mutation (N→D) is appropriate. It helps stabilize the negatively charged transition state by forming hydrogen bonds or neutralizing part of the charge. The suitable protonation balance not only alleviates the excessive desolvation effect at low pH, but also significantly improves electrostatic interactions by optimizing charge distribution. The addition of the new proton donor stabilizes the enzyme-substrate complex by stabilizing the hydrophobic core and enhancing hydrophobic interactions. This results in the KERQ7-123D mutant having a protonation and charge distribution that is more adapted to low pH environments, thereby improving its stability and activity in acidic environments.

[0100] Example 3: Investigation into the effect of the local stability of oxygen negative ion holes in KERQ7-123D on acid resistance

[0101] This example demonstrates how adding hydrophobic interactions to the KERQ7-123D oxon hole mutant from Example 2 achieves local stability, resulting in the mutant Q7-152V. Molecular dynamics simulations of KERQ7-123D-AAPF at pH 3 and 5 show that the RMSD of the KERQ7-123D-AAPF complex framework at pH 3 is significantly higher than that at pH 4 and 5, suggesting instability at pH 3. Therefore, this mutation introduces hydrophobic interactions to maintain the local conformation, further enhancing the activity of the KERQ7-123D mutant at pH 3. The distances to the key amino acids Ser221 and Ser125 of the oxon hole were investigated. The amino acid residues were ultimately determined to be at a distance of [missing information] from both Ser221 and Ser125. Ala152 within (distance from Ser221) Distance from Ser125 The amino acid Val was site-directed to induce hydrophobic forces in the oxygen anion hole to maintain the conformation.

[0102] (1) Using the shuttle plasmid DH5α-pHS-cotG-KERQ7-123D in step (1) of Example 2 as a template, the DH5α-pHS-cotG-KERQ7-123D plasmid was amplified by one-step PCR using primers 152V-F and 152V-R, and finally the mutant plasmid was obtained, which was denoted as pHS-cotG-KERQ7-152V;

[0103] The PCR amplification conditions were: 94℃ for 2 min, 98℃ for 10 s, 63℃ for 30 s, 68℃ for 2.5 min, for 30 cycles; the sequence of 152V-F was 5'-gttgttgttgttgcagtagcgggaaatagcggc-3'; the sequence of 152V-R was 5'-atttcccgctactgcaacaacaacaacgcc-3'. The KERQ7-152V keratinase mutant fragment in the mutant plasmid pHS-cotG-KERQ7-152V was designated as KERQ7-152V; the nucleotide sequence of KERQ7-152V is shown in SEQ ID NO.7, and its encoded amino acid sequence is shown in SEQ ID NO.8; finally, pHS-cotG-KERQ7-152V was transformed into E. coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-152V was extracted.

[0104] (2) Using the amino acid sequence of the keratinase mutant fragment KERQ7-152V from step (1) as a template, Alphafold2 was selected to construct a three-dimensional protein model, which was then denoted as Q7-152V. Subsequently, Q7-152V was molecularly docked with the keratin analog AAPF (N-succinyl-ala-ala-pro-phe-p-nitroanilide, PubChem CID5496888) to obtain the Q7-152V-AAPF protein-ligand complex. Molecular dynamics simulations were performed on this complex in an acidic pH environment (pH=3-5), and the three-dimensional protein structure model at pH=3 was selected and denoted as 152V. The trajectory files obtained from the molecular dynamics simulation of Q7-152V-AAPF, including root mean square deviation (RMSD) and root mean square fluctuation (RMSF), were analyzed using VMD 1.9.3. The visualization of the static structure and function of Q7-N-AAPF was performed using Pymol 2.5.5. The gmx_mmpbsa tool was used to extract stationary trajectory data from molecular dynamics simulations for MM-PBSA calculations.

[0105] This example describes the mutation process from KERQ7-123D to KERQ7-152V, and compares the acid stability and activity of the keratinase mutant B. subtilis cotG-KERQ7-123D with the introduction of negative charge by the oxy-ion hole in Example 2 with the keratinase mutant B. subtilis cotG-KERQ7-152V with locally increased hydrophobic interaction by the oxy-ion hole.

[0106] The spore enzyme preparation B. subtilis DB403 cotG-KERQ7-A152V was prepared according to the method for preparing keratinase enzyme preparation in step (5) of Example 1, the only difference being that the complete plasmid DH5α-pHS-cotG-KERQ7-N was replaced with DH5α-pHS-cotG-KERQ7-152V, and the keratinase enzyme preparation was finally obtained, which was denoted as B. subtilis DB403 cotG-KERQ7-A152V;

[0107] Using the same method for determining the degradation activity of feather meal as in Example 1, the changes in the degradation activity of B. subtilis DB403 cotG-KERQ7-123D and B. subtilis DB403 cotG-KERQ7-A152V (labeled A152V in the figure) prepared in Example 2 were compared in different pH (3-5) environments.

[0108] from Figure 7 The results show that, after introducing valine to supplement hydrophobic interaction, the activity of the KERQ7-152V mutant was significantly reduced compared to the KERQ7-123D mutant. This phenomenon will be analyzed and discussed below. First, molecular dynamics simulations of the KERQ7-152V-AAPF complex were performed at pH = 3, 4, and 5 for 50 ns. The structure of the KERQ7-152V-AAPF complex after a 50 ns MD simulation at pH = 3 was derived and observed. It was found that under these conditions, no hydrogen bonds were formed between Ser221 and the substrate or other amino acid residues. Figure 8 Furthermore, comparing the MD simulation results of the 152V and M3 complexes at pH=3, the RMSF fluctuation in the region near the catalytic residue Ser221 was relatively large. Figure 9 The hydrophobic interaction of valine locks in key regions, inhibiting conformational changes in the substrate. These phenomena essentially indicate that the significant decrease in activity is due to the strong hydrophobic side chain of Val disrupting local polarity, and the β-carbon branching structure of Val compressing the catalytic dimer, leading to catalytic residue misalignment. The distance between Glu64 and Ser221 in the catalytic dimer increases, causing the hydrogen bond network at the catalytic site to fail. This also suggests that the linear hydrophobic residue Ala before the mutation has better spatial compatibility. The results show that the protonated state of the KERQ7-123D mutant can ensure the stability of the complex in low pH environments; the addition of hydrophobic interactions alone would be counterproductive.

[0109] Conclusion: In the study of the oxygen anion stabilization mechanism during peptide bond formation in ribosome-synthesized proteins, it was found that ribosomes provide minimal transition state charge stabilization for oxygen anions during peptide bond formation via water molecules. This is also a mechanism of proton shuttle neutralization of transition state charge, thereby stabilizing the transition state. However, this mechanism differs from that of serine proteases, which typically exhibit more significant oxygen anion stabilization, such as stabilizing negatively charged transition states through specific hydrogen bonds or structures. Therefore, this section modifies the oxyanion pore chemistry of the keratinase KERQ7 by rationally designing the direct introduction of acidic amino acid residues into the oxygen anion pore. This requires computational simulation and synergistic optimization of the hydrogen bond network and proton transfer pathway, enabling the KERQ7 mutant to more actively and spontaneously regulate charge density through pH-dependent protonation. This mechanism enhances the acid resistance of serine proteases, ultimately achieving efficient and stable enzyme catalytic function under acidic conditions.

[0110] Example 4: The effect of spore-forming enzyme preparation on digesting feather meal in an acidic gastric environment

[0111] The stomach of monogastric animals is typically an acidic environment; the pH of porcine gastric juice is usually around 2-4. Feather meal has a high acid-binding capacity; adding feather meal to simulated gastric juice can raise its pH to around 6.2.

[0112] Simulated gastric fluid (SGF) was prepared according to the United States Pharmacopeia (2000) (2g NaCl, 3.2g pepsin (containing 800-1500 active units per mg) dissolved in 7.0mL hydrochloric acid to 1L, at which point the pH of the solution was 1.2).

[0113] Because feather meal has a strong acid-binding capacity, it takes a long time for the gastric juice environment of pigs to return to its normal pH of 1.2 after ingestion. Furthermore, in actual digestion, the pH of animal gastric juice does not usually remain at 1.2. Studies by Kopper et al. showed that soluble peanut protein was undetectable 1 hour after consuming peanut meal, and the stomach pH only dropped to between 2 and 3 after 0.75 hours. Therefore, in this case, feather meal was added to the prepared simulated gastric juice, and the pH was adjusted back to 3.5 before incubating the mixture.

[0114] This experiment consists of 5 groups, as follows:

[0115] (1) Undigested: Feather powder;

[0116] (2) Digested without keratinase: Add 3g of feather meal to 100mL of artificial gastric juice and adjust the pH value to 3.5;

[0117] (3) Digested with 15% Ding-S spore preparation: 3g of feather meal was added to 100mL of artificial gastric juice, and 15% of B. subtilis DB403 cotG-KERQ7-Ding-S enzyme preparation was added (i.e., the ratio of enzyme preparation to feather meal was 15ml:100g), and the pH value was adjusted to 3.5.

[0118] (4) Digested with 15% T2 spore preparation: 3g of feather meal was added to 100mL of artificial gastric fluid, and 15% of B. subtilis DB403cotG-KERQ7-T2 enzyme preparation was added (i.e., the ratio of enzyme preparation to feather meal was 15ml:100g), and the pH value was adjusted to 3.5.

[0119] (5) Digested with 20% T2 spore preparation: 3g of feather meal was added to 100mL of artificial gastric fluid, and 20% of B. subtilis DB403cotG-KERQ7-T2 enzyme preparation was added (i.e., the ratio of enzyme preparation to feather meal was 20ml:100g), and the pH value was adjusted to 3.5.

[0120] All five groups were incubated at 37℃ and 95rpm for 1 hour, then dried to constant weight, and the crude protein and small peptide contents were determined according to the national standard GB / T22492-2008.

[0121] Figure 10 This study investigated the effect of enzyme preparations on the content of small peptides produced by the degradation of feather meal feed in an artificial gastric juice environment. The results showed that the small peptide content in the untreated digestion group was only 3.75±0.15%, which was not significantly different from the 3.19±0.055% in the untreated group. After adding 15% (mL / g) of *B. subtilis* DB403 cotG-KERQ7-S enzyme preparation, the post-digestion small peptide content increased to 7.71±0.26%. Adding 15% (mL / g) of *B. subtilis* DB403 cotG-KERQ7-T2 enzyme preparation increased the post-digestion small peptide content to 10.85±0.36%, and adding 20% ​​(mL / g) of *B. subtilis* DB403 cotG-KERQ7-T2 enzyme preparation significantly increased the post-digestion small peptide content to 17.30±0.18%.

[0122] Combining the previous data and conclusions, it is clear that the low digestibility and high acid-binding capacity of feather meal explain why it has been considered a low-quality feed for decades. The key to large-scale utilization of feather meal resources in the feed industry lies in improving its palatability and digestibility, and modifying keratinase according to the capabilities of industrial feed applications to enhance its acid resistance and specific activity in acidic environments, thereby increasing its palatability and the proportion of digestible protein.

[0123] Example 5: Spore demonstration of the effect of keratinase solid powder on the digestion of feather meal in an acidic gastric environment

[0124] The B. subtilis DB403 cotG-KERQ7-T2 enzyme preparation (suspension) can be freeze-dried to obtain a solid powder. Based on Example 4, the difference is that the suspension (B. subtilis DB403 cotG-KERQ7-T2) is replaced with a solid powder. Specifically, the keratinase preparation B. subtilis DB403 cotG-KERQ7-T2 is added to the fermentation system in the form of a freeze-dried solid powder. Specifically, 3g of feather meal is added to 100mL of artificial gastric juice, and the addition amount is 15% of the feather meal (i.e., the ratio of enzyme preparation to feather meal is 15g:100g). Other conditions remain unchanged, and the test is performed according to the method of Example 4.

[0125] The final measured content of small peptides was 17.09±0.06%, which was not significantly different from the group in Example 4 with 15% spore suspension enzyme preparation B. subtilis DB403 cotG-KERQ7-T2. This indicates that the addition of spore enzyme preparation in solid powder form is equally efficient and feasible, and can achieve significant technical effects.

[0126] 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 the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

[0127] Sequence Listing SEQ ID NO.1

[0128] The nucleotide sequence of the mature fragment of Ding-S: GCTCAGACAGTGCCGTATGGACAGGAACAGATTAAGGATCCGGCAGTGGAGGCGCAAGGATATAAAGGAGCAAATGTGAAAGTGGCAGTTCTGGATACAGGCATTGAGGCAGCACATCCGGATCTGAATGTTGCAGGAGGCGCGTCATTTGCGCCGTCAGAACCGAATGCGACACAAGATTTTCAATCAGAGGCACAACATGTTGCAGGAACAATTGCAGCACTTGATAATACAATTGGAGTTCTGGGCGTGGCACCGAGCGCAAGCCTGTATGCGGTGAAAGTTCTTGATGAGAATGGAGATGGCCAATATTCATGGATTATTTCAGGCATTGAATGGGCGGTGGCAAATAATATGGATGTTATTAATATGAGCCTGGGCGGACCGAATGGCAGCACAGCGCTTAAAAATGCGGTGGATACAGCGAATAATGAGGGCGTTGTTGTTGTTGCAGCAGCGGGAAATAGCGGCAGCTTTGGCAGCACATCAACAGTGGGATATCCGGCAAAATATGATTCAACAGGAGCGGTTGCAAATGTGAATTCAAATAATGTGAACAACAGCAGCAGCAGCGCGGGACCGGAACTGGATGTTAGCGCGCCGGGCACAAGCATTCTGAGCACAGTTCCGTCATCAGGATATACATCATATACAGCGACATCAATGGCATCACCGCATGTTGCGGGAGCAGCAGCGGTTATTCTTTCAAAATATCCGAATCTGACAAACAGCCAGGTTCGCCAGCAACTGGAAAATACAGCAACACCGCTTGGCGATAGCTTTTATTATGGCAAAGGACTTATTAACGTCCAGGCGGCGAGCAATTAA

[0129] SEQ ID NO.2

[0130] The amino acid sequence encoded by the mature fragment of Ding-S is AQTVPYGQEQIKDPAVEAQGYKGANVKVAVLDTGIEAAHPDLNVAGGASFAPSEPNATQDFQSEAQHVAGTIAALDNTIGVLGVAPSASLYAVKVLDENGDGQYSWIISGIEWAVANNMDVINMSLGGPNGSTALKNAVDTANNEGVVVVAAAGNSGSFGSTSTVGYPAKYDSTGAVANVNSNNVNNSSSSAGPELDVSAPGTSILSTVPSSGYTSYTATSMASPHVAGAAAVILSKYPNLTNSQVRQQLENTATPLGDSFYYGKGLINVQAASN

[0131] SEQ ID NO.3

[0132] KERQ7-N nucleotide sequence

[0133] GCTCAGACAGTGCCGTATGGACAGGAACAGATTAAGGATCCGGCAGTGGAGGCGCAAGGATATAAAGGAGC

[0134] AAATGTGAAAGTGGCAGTTCTGGATACAGGCATTGAGGCAGCACATCCGGATCTGAATGTTGCAGGAGGCGC

[0135] GTCATTTGCGCCGTCAGAACCGAATGCGACACAAGATTTTCAATCAGAGGCACAACATGTTGCAGGAACAAT

[0136] TGCAGCACTTGATAATACAATTGGAGTTCTGGGCGTGGCACCGAGCGCAAGCCTGTATGCGGTGAAAGTTCT

[0137] TGATGAGAATGGAGATGGCCAATATTCATGGATTATTTCAGGCATTGAATGGGCGGTGGCAAATAATATGGATG

[0138] TTATTAATATGAGCCTGGGCGGACCGAATGGCAGCACAGCGCTTAAAAATGCGGTGGATACAGCGAATAATGA

[0139] GGGCGTTGTTGTTGTTGCAGCAGCGGGAAATAGCGGCAGCTTTGGCAGCACATCAACAGTGGGATATCCGGC

[0140] AAAATATGATTCAACAGGAGCGGTTGCAAATGTGAATTCAAATAATGTGAACAACAGCAGCAGCAGCGCGGG

[0141] ACCGGAACTGGATGTTAGCGCGCCGGGCACAAGCATTCTGAGCACAGTTCCGTCATCAGGATATACATCATAT

[0142] ACAGCGACATCAATGGCATCACCGCATGTTGCGGGAGCAGCAGCGGTTATTCTTTCAAAATATCCGAATCTGA

[0143] CAAACAGCCAGGTTGAGCAGCAACTGGAAAATACAGCAACACCGCTTGGCGATAGCTTTTATTATGGCAAAG

[0144] GACTTATTAACGTCCAGGCGGCGAGCAATTAA

[0145] SEQ ID NO.4

[0146] KERQ7-N encoded amino acid sequence AQTVPYGQEQIKDPAVEAQGYKGANVKVAVLDTGIEAAHPDLNVAGGASFAPSEPNATQDFQSEAQHVAGTIAALDNTIGVLGVAPSASLYAVKVLDENGDGQYSWIISGIEWAVANNMDVINMSLGGPNGSTALKNAVDTANNEGVVVVAAAGNSGSFGSTSTVGYPAKYDSTGAVANVNSNNVNNSSSSAGPELDVSAPGTSILSTVPSSGYTSYTATSMASPHVAGAAAVILSKYPNLTNSQVEQQLENTATPLGDSFYYGKGLINVQAASN

[0147] SEQ ID NO.5

[0148] KERQ7-123D nucleotide sequence: GCTCAGACAGTGCCGTATGGACAGGAACAGATTAAGGATCCGGCAGTGGAGGCGCAAGGATATAAAGGAGCAAATGTGAAAGTGGCAGTTCTGGATACAGGCATTGAGGCAGCACATCCGGATCTGAATGTTGCAGGAGGCGCGTCATTTGCGCCGTCAGAACCGAATGCGACACAAGATTTTCAATCAGAGGCACAACATGTTGCAGGAACAATTGCAGCACTTGATAATACAATTGGAGTTCTGGGCGTGGCACCGAGCGCAAGCCTGTATGCGGTGAAAGTTCTTGATGAGAATGGAGATGGCCAATATTCATGGATTATTTCAGGCATTGAATGGGCGGTGGCAAATAATATGGATGTTATTGATATGAGCCTGGGCGGACCGAATGGCAGCACAGCGCTTAAAAATGCGGTGGATACAGCGAATAATGAGGGCGTTGTTGTTGTTGCAGCAGCGGGAAATAGCGGCAGCTTTGGCAGCACATCAACAGTGGGATATCCGGCAAAATATGATTCAACAGGAGCGGTTGCAAATGTGAATTCAAATAATGTGAACAACAGCAGCAGCAGCGCGGGACCGGAACTGGATGTTAGCGCGCCGGGCACAAGCATTCTGAGCACAGTTCCGTCATCAGGATATACATCATATACAGCGACATCAATGGCATCACCGCATGTTGCGGGAGCAGCAGCGGTTATTCTTTCAAAATATCCGAATCTGACAAACAGCCAGGTTGAGCAGCAACTGGAAAATACAGCAACACCGCTTGGCGATAGCTTTTATTATGGCAAAGGACTTATTAACGTCCAGGCGGCGAGCAATTAA

[0149] SEQ ID NO.6

[0150] KERQ7-123D encoded amino acid sequence AQTVPYGQEQIKDPAVEAQGYKGANVKVAVLDTGIEAAHPDLNVAGGASFAPSEPNATQDFQSEAQHVAGTIAALDNTIGVLGVAPSASLYAVKVLDENGDGQYSWIISGIEWAVANNMDVIDMSLGGPNGSTALKNAVDTANNEGVVVVAAAGNSGSFGSTSTVGYPAKYDSTGAVANVNSNNVNNSSSSAGPELDVSAPGTSILSTVPSSGYTSYTATSMASPHVAGAAAVILSKYPNLTNSQVEQQLENTATPLGDSFYYGKGLINVQAASN

[0151] SEQ ID NO.7

[0152] KERQ7-152V nucleotide sequence: GCTCAGACAGTGCCGTATGGACAGGAACAGATTAAGGATCCGGCAGTGGAGGCGCAAGGATATAAAGGAGCAAATGTGAAAGTGGCAGTTCTGGATACAGGCATTGAGGCAGCACATCCGGATCTGAATGTTGCAGGAGGCGCGTCATTTGCGCCGTCAGAACCGAATGCGACACAAGATTTTCAATCAGAGGCACAACATGTTGCAGGAACAATTGCAGCACTTGATAATACAATTGGAGTTCTGGGCGTGGCACCGAGCGCAAGCCTGTATGCGGTGAAAGTTCTTGATGAGAATGGAGATGGCCAATATTCATGGATTATTTCAGGCATTGAATGGGCGGTGGCAAATAATATGGATGTTATTGATATGAGCCTGGGCGGACCGAATGGCAGCACAGCGCTTAAAAATGCGGTGGATACAGCGAATAATGAGGGCGTTGTTGTTGTTGCAGTTGCGGGAAATAGCGGCAGCTTTGGCAGCACATCAACAGTGGGATATCCGGCAAAATATGATTCAACAGGAGCGGTTGCAAATGTGAATTCAAATAATGTGAACAACAGCAGCAGCAGCGCGGGACCGGAACTGGATGTTAGCGCGCCGGGCACAAGCATTCTGAGCACAGTTCCGTCATCAGGATATACATCATATACAGCGACATCAATGGCATCACCGCATGTTGCGGGAGCAGCAGCGGTTATTCTTTCAAAATATCCGAATCTGACAAACAGCCAGGTTGAGCAGCAACTGGAAAATACAGCAACACCGCTTGGCGATAGCTTTTATTATGGCAAAGGACTTATTAACGTCCAGGCGGCGAGCAATTAA

[0153] SEQ ID NO.8

[0154] The amino acid sequence encoded by KERQ7-152V is AQTVPYGQEQIKDPAVEAQGYKGANVKVAVLDTGIEAAHPDLNVAGGASFAPSEPNATQDFQSEAQHVAGTIAALDNTIGVLGVAPSASLYAVKVLDENGDGQYSWIISGIEWAVANNMDVIDMSLGGPNGSTALKNAVDTANNEGVVVVAVAGNSGSFGSTSTVGYPAKYDSTGAVANVNSNNVNNSSSSAGPELDVSAPGTSILSTVPSSGYTSYTATSMASPHVAGAAAVILSKYPNLTNSQVEQQLENTATPLGDSFYYGKGLINVQAASN.

Claims

1. A keratinase mutant that efficiently degrades feather feed in an acidic gastric environment, characterized in that, The keratinase mutant, KERQ7-123D, is formed by mutating arginine (Arg) at position 247 to glutamic acid (Glu) and asparagine (Asn) at position 123 to aspartic acid (Asp) in the keratinase Ding-S mutant sequence shown in SEQ ID NO.

2. The nucleotide sequence of the keratinase Ding-S mutant is shown in SEQ ID NO.

1. The method for preparing the keratinase mutant KERQ7-123D is as follows: (1) Using the amino acid sequence SEQ ID NO.2 of the keratinase mutant fragment Ding-S as a template, a three-dimensional protein model was constructed, and the resulting protein model was denoted as Q7-S. Subsequently, Q7-S was molecularly docked with the keratin analog AAPF to obtain the Q7-S-AAPF protein ligand complex. Molecular dynamics simulations of the Q7-S-AAPF protein ligand complex were performed in an acidic pH environment. The root mean square fluctuation 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 trend line of the root mean square deviation, with lower values ​​indicating greater stability. The most stable protein three-dimensional structure model was selected and denoted as M1. Based on the root mean square fluctuation values ​​of each amino acid site in model M1, modification sites were selected. The modification site is the surface amino acid Arg247. The mature mutant fragment obtained by mutating Arg247Glu on the keratinase mutant fragment Ding-S is denoted as KERQ7-N. The nucleotide sequence of KERQ7-N is shown in SEQ ID NO.3, and the amino acid sequence it encodes is shown in SEQ ID NO.

4. (2) Using the amino acid sequence of KERQ7-N in step (1) as a template, a three-dimensional protein model was constructed, and the protein model was denoted as Q7-N. Then, Q7-N was molecularly docked with the keratin analog AAPF to obtain the Q7-N-AAPF protein ligand complex. Molecular dynamics simulation of the Q7-N-AAPF protein ligand complex was performed in an acidic pH environment. The stable three-dimensional structure of the Q7-N-AAPF protein ligand complex after equilibrium was obtained through molecular dynamics simulation. The stable three-dimensional protein structure model was denoted as M2. Based on the direct interaction network of the key component amino acid Ser125 of the oxygen negative ion hole in model M2 as a reference, the site modification was selected that is related to the composition of the oxygen negative ion hole but does not directly interact with it, i.e., the secondary interaction mode. The selected modification site was amino acid Asn123Asp. The mutant obtained after modification was KERQ7-123D. The obtained KERQ7-123D mutant is the keratinase mutant that efficiently degrades feather feed in an acidic gastric juice environment.

2. The keratinase mutant for efficiently degrading feather feed in an acidic gastric environment according to claim 1, characterized in that, In step (1), Alphafold2 was selected for constructing the three-dimensional protein model; Q7-S was docked with the keratin analog AAPF molecule through Autodock, and the docking posture was selected as the conformation in which the catalytic amino acid Ser221 of Q7-S directly interacts with the amide bond of AAPF. In the molecular dynamics simulation, the acidic pH environment is 4-6, and the pH environment corresponding to the most stable protein three-dimensional structure model is 4.

3. The keratinase mutant for efficiently degrading feather feed in an acidic gastric environment according to claim 1, characterized in that, In step (2), Alphafold2 was selected for constructing the three-dimensional protein model; Q7-N was docked with the keratin analog AAPF molecule through Autodock, and the docking posture was selected as the conformation in which the catalytic amino acid Ser221 of Q7-N directly interacts with the amide bond of AAPF. In the molecular dynamics simulation, the acidic pH environment is 3-5, while the pH environment corresponding to the stable protein three-dimensional structure model is 3.

4. A mutant gene encoding a keratinase that efficiently degrades feather feed in the acidic gastric juice environment, characterized in that, The sequence of the keratinase mutant gene encoding the efficient degradation of feather feed in the acidic gastric juice environment is shown in SEQ ID NO.5; The method for obtaining the keratinase mutant gene that efficiently degrades feather feed in an acidic gastric juice environment is as follows: (1) First, the KERQ7 mutant Ding-S, which is displayed on the surface of the anchoring protein cotG, was selected and denoted as cotG-KERQ7-S. The nucleotide sequence of the mature fragment of Ding-S is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.

2. Then, cotG-KERQ7-S was integrated into the Escherichia coli-Bacillus subtilis shuttle expression vector pHS to form the shuttle plasmid pHS-cotG-KERQ7-S. (2) Using the shuttle plasmid pHS-cotG-KERQ7-S described in step (1) as a template, a one-step whole plasmid PCR site-directed mutagenesis was performed to change the surface amino acid Arg247 to Glu247. The resulting mutant plasmid was named pHS-cotG-KERQ7-N, and the keratinase mutant fragment in the mutant plasmid pHS-cotG-KERQ7-N was named KERQ7-N. The nucleotide sequence of KERQ7-N is shown in SEQ ID NO.3, and the amino acid sequence it encodes is shown in SEQ ID NO.

4. Finally, pHS-cotG-KERQ7-N was transformed into Escherichia coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-N was extracted. (3) Using the DH5α-pHS-cotG-KERQ7-N obtained in step (2) as a template, a one-step whole plasmid PCR site-directed mutagenesis was performed to change the amino acid Asn123, which directly interacts with the key amino acid Ser125 of the oxygen negative ion hole, to Asp123. The resulting mutant plasmid was named pHS-cotG-KERQ7-123D. The keratinase mutant fragment in the mutant plasmid pHS-cotG-KERQ7-123D was named KERQ7-123D. The gene sequence encoding KERQ7-123D was obtained as shown in SEQ ID NO.

5.

5. The keratinase mutant gene encoding efficient degradation of feather feed in an acidic gastric environment according to claim 4, characterized in that, The steps for obtaining the shuttle plasmid pHS-KERQ7-S in step (1) are as follows: Using the sequence SEQ ID NO.1 of Ding-S as a template, Ding-S is amplified with primers Q7-F and Q7-R to obtain the amplified Ding-S fragment. Then, the Ding-S fragment is inserted into the Escherichia coli-Bacillus subtilis shuttle expression vector pHS-cotG plasmid using Spe I and Hind III restriction enzymes to construct the recombinant shuttle plasmid, denoted as pHS-cotG-KERQ7-S. The amplification conditions are: 94℃ for 3 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, for 30 cycles. The sequence of Q7-F is 5'-ggactagtgctcagacagtgccgtatgg-3'. The sequence of Q7-R is 5'-cccaagcttttaattgctcgccgcctgga-3'. The specific steps for obtaining pHS-cotG-KERQ7-N in step (2) are as follows: using the shuttle plasmid pHS-cotG-KERQ7-S as a template, the pHS-cotG-KERQ7-S plasmid is amplified by one-step PCR using primers 247E-F and 247E-R, and finally the mutant plasmid is obtained, which is denoted as pHS-cotG-KERQ7-N; The PCR amplification conditions were: 94℃ for 2 min, 98℃ for 10 s, 63℃ for 30 s, 68℃ for 2.5 min, for 30 cycles; the sequence of 247E-F was 5'-cagccaggttcgccagcaactggaaaatacagc-3'; the sequence of 247E-R was 5'-ccagttgctggcgaacctggctgtttgtcag-3'; The steps for obtaining pHS-cotG-KERQ7-123D in step (3) are as follows: Using plasmid DH5α-pHS-cotG-KERQ7-N as a template, the DH5α-pHS-cotG-KERQ7-N plasmid is amplified by one-step PCR using primers 123D-F and 123D-R, and finally the mutant plasmid is obtained, which is denoted as pHS-cotG-KERQ7-123D; The PCR amplification conditions were: 94℃ for 3 min, 98℃ for 10 s, 63℃ for 30 s, 68℃ for 2.5 min, for 30 cycles; The primer sequence for 123D-F is 5'-atgttattgatatgagcctgggcggaccgaatg-3'; the primer sequence for 123D-R is 5'-cgcccaggctcatatcaataacatccatattatttgcc-3'.

6. An engineered bacterium expressing a keratinase mutant that efficiently degrades feather feed in an acidic gastric juice environment, characterized in that, The keratinase mutant engineered bacteria comprises the expression vector pHS-cotG-KERQ7-123D; the expression vector pHS-cotG-KERQ7-123D comprises the keratinase mutant gene encoding the efficient degradation of feather feed in an acidic gastric environment as described in claim 4; the keratinase mutant engineered bacteria are obtained by introducing the expression vector pHS-cotG-KERQ7-123D into host cells. The steps are as follows: pHS-cotG-KERQ7-123D is transformed into Escherichia coli DH5α, and the complete plasmid DH5α-pHS-cotG-KERQ7-123D is extracted; using Bacillus subtilis DB403 as the host bacterium, the recombinant shuttle plasmid DH5α-pHS-cotG-KERQ7-123D is introduced by electroporation to construct the recombinant engineered bacterium B. subtilis DB403 pHS-cotG-KERQ7-123D.

7. A method for preparing a mutant enzyme preparation, characterized in that, Based on the recombinant engineered bacteria described in claim 6, after inoculation into a fermentation medium and cultivation, a recombinant spore protein suspension with keratinase on its surface is obtained by centrifugation; or the recombinant spore protein suspension is freeze-dried to obtain a solid powder; the recombinant spore protein suspension or solid powder is a keratinase preparation that is highly efficient in degrading feather feed in an acidic gastric environment; the keratinase preparation is a mutant enzyme preparation, denoted as B. subtilisDB403 cotG-KERQ7-T2.

8. The method for preparing the mutant enzyme preparation according to claim 7, characterized in that, The engineered strain *B. subtilis* DB403pHS-cotG-KERQ7-123D was inoculated into LB medium and cultured for the first time to obtain a culture broth. The culture broth was then inoculated into DSM liquid medium, cultured for the second time, and centrifuged to collect the bacterial precipitate. The precipitate was resuspended in GTE buffer containing lysozyme. The resuspended precipitate was incubated, centrifuged again, and washed with PBS buffer. The precipitate was then resuspended in PBS buffer to obtain a recombinant spore protein suspension exhibiting keratinase on its surface. Alternatively, the recombinant spore protein suspension was freeze-dried to obtain a solid powder. Both the recombinant spore protein suspension and the solid powder are keratinase preparations, designated as *B. subtilis* DB403 cotG-KERQ7-T2. The concentration of lysozyme in the GTE Buffer is 2 mg / mL, and the components of the GTE Buffer are: 10 mM EDTA, 20 mM Tris-HCl (pH = 7.5), and 50 mM Glucose. The first culture conditions were 37℃, 200rpm overnight; the second culture conditions were 37℃, 200rpm, 36h; the incubation conditions were 37℃, 60min, the pH of the PBS buffer was 7.4, and the composition of the PBS buffer was as follows (per 1L): 8g NaCl, 0.2g KCl, 1.42g Na2HPO4, 0.27g KH2PO4; the OD600nm of the recombinant spore protein suspension was 1-2.

9. The mutant enzyme preparation prepared according to the method of claim 7 or 8 is used for the degradation of feather meal in acidic gastric juice.

10. The use according to claim 9, characterized in that, The steps are as follows: (1) Prepare artificial gastric juice, based on 1L: NaCl 2g; pepsin 3.2g, each mg of pepsin contains 800-1500 active units; hydrochloric acid 7mL; dissolve in deionized water and bring the volume to 1L, the pH of the solution is 1.2; (2) Add keratinase preparation and feather powder to artificial gastric juice and adjust the pH to obtain a mixture. After incubation, the mixture can be used to efficiently degrade feather feed in an acidic gastric juice environment. The ratio of feather meal to artificial gastric fluid is 3g:100mL; when the keratinase preparation is a recombinant spore protein suspension, the addition amount is 15%-20% of the total feather meal, i.e., 15-20ml:100g; when the keratinase preparation is a solid powder, the addition amount is 15%-20% of the total feather meal, i.e., 15-20g:100g; the pH is adjusted to 3.5; the incubation conditions are: 37℃, 95rpm for 1h.

Images