Cutinase mutant for improving catalytic efficiency of polyvinyl acetate and application of cutinase mutant

Abstract

The invention discloses a cutinase mutant with remarkably enhanced catalytic efficiency on polyvinyl acetate and application of the cutinase mutant, and belongs to the technical field of enzyme engineering and genetic engineering. The combined mutant C11 provided by the invention shows that the catalytic efficiency on polyvinyl acetate is obviously improved, and the yield of a degradation product acetic acid is 1.73 times that of M8. In addition, the half-life period of C11 at 70 DEG C is 205.9 hours, and the half-life period is prolonged by 5.17 times compared with the half-life period of M8. Meanwhile, the stability of the C11 in an alkaline environment is enhanced, so that the C11 has higher alkali resistance, and the application potential of the C11 under an alkaline condition is widened. The cutinase mutant provided by the invention represents a biocatalyst which has a great prospect in application of industrial paper pulp and paper.

Description

Technical Field

[0001] This invention relates to a keratinase mutant that significantly enhances the catalytic efficiency of polyvinyl acetate and its applications, belonging to the fields of enzyme engineering and genetic engineering technology. Background Technology

[0002] In the papermaking process, polyvinyl acetate (PVAc) compounds are a major component of adhesive contaminants in the white water system. These substances easily accumulate on the surfaces of large equipment in the papermaking industry, negatively impacting heat transfer efficiency and causing physical defects such as spots and holes in the produced paper, severely affecting the quality of the paper product. Conventional chemical treatment methods require the continuous addition of cationic flocculants to neutralize PVAc compounds in the white water. While this can temporarily alleviate the deposition of sticky substances, it also increases treatment costs, reduces pulp quality due to residual chemicals, and generates environmental risks such as organochlorine byproducts.

[0003] Keratinase (EC 3.1.1.74), an ester-linked hydrolase belonging to the α / β hydrolase family, can act on a variety of compounds containing ester bonds due to its unique active site structure. Notably, this enzyme also exhibits hydrolytic potential against synthetic polyester substrates, providing a theoretical basis for developing biological treatment methods for paper adhesives. Existing research indicates that... Humicola insolens and Fusarium solani pisi While natural keratinases from microorganisms possess PVAc hydrolytic capabilities, their catalytic efficiency is generally below the threshold for industrial applications. More importantly, pulping and papermaking processes require enzyme preparations to maintain stable activity under high temperature and alkaline conditions, such as pH 8.0 and 60℃. Wild-type keratinases are prone to conformational collapse in this environment, resulting in a sharp decline in catalytic function and insufficient catalytic activity for synthetic polyesters.

[0004] Protein engineering modification strategies are effective methods to improve enzyme catalytic performance. By rationally or irrationally modifying enzyme molecules through techniques such as point mutation and directed evolution, their catalytic activity, stability, or substrate specificity can be improved. Studies have reported on the modification of keratinases, for example... Thermobifida fuscaModifying residues near the active pocket of keratinase can enhance its degradation efficiency against polyethylene terephthalate (PET). Current research primarily focuses on the degradation of PET plastics, with few reports on mutants that improve the catalytic efficiency of keratinase against polyvinyl acetate (PVAc). However, PVAc and PET differ fundamentally in their chemical structures. PVAc is a carbon-chain polymer formed by the free radical polymerization of vinyl acetate monomers, with acetoxy groups on its side chains, while PVAc is an aromatic polyester linked by ester bonds. This difference means that highly efficient PET-degrading enzymes may be completely ineffective against PVAc. Currently, there are few systematic studies in the published literature on the targeted modification of keratinase to address the molecular characteristics of PVAc, especially lacking engineered enzymes that exhibit both high activity and high stability in high-temperature, alkaline environments. Therefore, there is an urgent need in this field to develop a keratinase mutant with significantly enhanced catalytic efficiency against PVAc, capable of stably exerting catalytic activity in the alkaline, high-temperature environment of the pulp and paper industry. Summary of the Invention

[0005] Technical issues The technical problem to be solved by this invention is that wild-type keratinases generally have low catalytic efficiency for polyvinyl acetate, which makes it difficult to meet the actual application needs of the pulp and paper industry.

[0006] Technical solution The present invention provides a keratinase mutant, which is based on the starting sequence shown in SEQ ID NO.1 and has one or more mutations among T29G, T29A, T29L, T37S, T37V, L66P, ​​L66W, I168V, L174F or L174W.

[0007] In one embodiment, the mutant is a mutant T29G, T29A, or T29L obtained by mutating threonine at position 29 to glycine, alanine, or leucine.

[0008] In one embodiment, the mutant is T37S or T37V, obtained by mutating threonine at position 37 to serine or valine.

[0009] In one embodiment, the mutant is a mutant L66P or L66W obtained by mutating leucine at position 66 to proline or tryptophan.

[0010] In one embodiment, the mutant is mutant I168V obtained by mutating isoleucine at position 168 to valine.

[0011] In one embodiment, the mutant is a mutant L174F or L174W obtained by mutating leucine at position 174 to phenylalanine or tryptophan.

[0012] In one embodiment, the mutant is L174F / I168V, obtained by mutating leucine at position 174 to phenylalanine and isoleucine at position 168 to valine.

[0013] In one embodiment, the mutant is L174W / L66W, obtained by mutating leucine at position 174 to tryptophan and leucine at position 66 to tryptophan.

[0014] In one embodiment, the mutant is L174F / I168V, obtained by mutating leucine at position 174 to phenylalanine, isoleucine at position 168 to valine, and L174F / I168V / T29G, obtained by mutating threonine at position 29 to glycine.

[0015] The present invention also provides a gene encoding the mutant.

[0016] The present invention also provides a recombinant vector containing the said gene.

[0017] In one embodiment, the carrier includes, but is not limited to, pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series carriers.

[0018] The present invention also provides recombinant microorganisms expressing the mutants.

[0019] In one embodiment, the microorganisms include, but are not limited to, Escherichia coli, Bacillus subtilis, or Pichia pastoris.

[0020] In one embodiment, the recombinant microorganism is recombinant Escherichia coli.

[0021] In one embodiment, the recombinant Escherichia coli uses pET20b(+) as a vector, and... E. coli BL21(DE3)pLysS is the host.

[0022] The present invention also provides a method for preparing the keratinase mutant.

[0023] In one embodiment, the method includes fermenting and culturing the recombinant *E. coli* and isolating keratinase. The method involves culturing the recombinant *E. coli* in a culture medium and collecting the keratinase mutant from the culture medium.

[0024] The present invention also provides a method for improving the catalytic efficiency of keratinase, which involves site-directed mutagenesis of the keratinase with an amino acid sequence as shown in SEQ ID NO.1.

[0025] In one embodiment, the mutation includes one or more of the following mutations: T29G, T29A, T29L, T37S, T37V, L66P, ​​L66W, I168V, L174F, or L174W.

[0026] In one embodiment, the mutation is any combination of the following mutations: L174F / I168V, L174W / L66W, and L174F / I168V / T29G.

[0027] This invention provides the application of the keratinase mutant in the degradation of PVAc at a high temperature of 60°C and pH of 8.0.

[0028] In one embodiment, the application includes, but is not limited to, polyester degradation and detergent enhancement.

[0029] Beneficial effects Compared with the keratinase M8, the mutant of this invention has improved catalytic efficiency and thermal stability in degrading PVAc.

[0030] In PVAc degradation experiments, the single-point mutants T29G, T29A, T29L, T37S, T37V, L66P, ​​L66W, I168V, L174F (C9), or L174W showed increased acetic acid production compared to the keratinase-initiating enzyme M8 (11.47 mg / L), with increases ranging from 1.03 to 1.24 times. PVAc is an ester polymer, but its strong hydrophobicity, flexible molecular chains, and poor accessibility in aqueous phases pose significant challenges to enzymatic reactions primarily mediated by water. Therefore, the aforementioned single-point mutants achieving acetic acid production 1.03 to 1.24 times that of M8 represent a considerable improvement.

[0031] The engineered mutants L174F / I168V (C10), L174W / L66W, and L174F / I168V / T29G (C11) showed acetic acid production increases of 1.53, 1.40, and 1.73 times relative to keratinase M8, respectively, with acetic acid production of 17.57 mg / L, 16.07 mg / L, and 19.85 mg / L.

[0032] In thermal stability experiments, compared with the keratinase M8, the engineered hybrid mutant C11 had a longer half-life (t) at 70 °C. 1 / 2 It has some improvement over the M8, its t 1 / 2 The half-life was 205.9 h, a 5.17-fold increase compared to M8. Overall, the mutant exhibited enhanced catalytic efficiency and thermal stability, broadening its application potential for PVAc degradation under high-temperature conditions. Attached Figure Description

[0033] Figure 1 The image shows the SDS-PAGE gel electrophoresis analysis of purified M8 and its series of single-point mutants; M represents the molecular weight of the standard protein, and the numbers 1 to 11 are the starting keratinases M8, T29G, T29A, T29L, T37S, T37V, L66P, ​​L66W, I168V, L174F or L174W, respectively.

[0034] Figure 2 The image shows the SDS-PAGE gel electrophoresis analysis of the purified combined mutants; M represents the molecular weight of the standard protein, and numbers 1 to 3 are the iterative combined mutants L174F / I168V, L174W / L66W, and L174F / I168V / T29G, respectively.

[0035] Figure 3 This invention uses a structure comparison strategy to screen mutants that improve the performance of PVAc degradation.

[0036] Figure 4 This invention uses a virtual screening strategy to screen mutants that improve the performance of PVAc degradation.

[0037] Figure 5 This invention uses a Funclib-assisted prediction strategy to screen mutants that improve PVAc degradation performance.

[0038] Figure 6 This invention uses M8 / L174F as a template to screen for combined mutants that improve the degradation performance of PVAc.

[0039] Figure 7 This invention uses M8 / L174W as a template to screen for combined mutants that improve the degradation performance of PVAc.

[0040] Figure 8 This invention uses M8 / L174F / I168V as a template to screen for combined mutants that improve PVAc degradation performance.

[0041] Figure 9 This invention uses M8 / L174F / I168V / T29G as a template to screen for combined mutants that improve PVAc degradation performance.

[0042] Figure 10 The half-life of M8 and its series of combined mutants at 70°C is given.

[0043] Figure 11 This is the optimal temperature for M8 and its series of combined mutants of the present invention.

[0044] Figure 12 The optimal pH for M8 and its series of combined mutants of this invention.

[0045] Figure 13 This invention relates to the pH stability of M8 and its series of combined mutants. Detailed Implementation

[0046] 1. Experimental strains and expression vectors The host strains involved in the embodiments of this invention include: *Escherichia coli* JM109 (from Novagen), used for gene cloning operations; and *Escherichia coli* BL21(DE3) pLysS (purchased from Beijing TransGen Biotech Co., Ltd.), used as the recombinant protein expression host. Genetic engineering operations were performed using the pET-20b(+) expression vector (Novagen product) carrying the T7 strong promoter.

[0047] 2. Enzyme preparations and other biochemical reagents Antibiotics: Ampicillin (Amp) was provided by Shanghai Sangon Biotech Co., Ltd. Protein detection system: Medium molecular weight protein marker standards and SDS-PAGE gel electrophoresis kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Enzyme activity assay substrate: p-Nitrophenylbutyrate ( p -nitrophenyl butyrate, p Both NPB and polyvinyl acetate (PVAc) are sourced from Sigma-Aldrich. Molecular biology reagents: PCR amplification primers were synthesized and purified by Suzhou Genewiz Biotechnology Co., Ltd. Metabolite detection: Quantitative analysis of acetic acid was performed using a dedicated kit from Megazyme. Common chemical reagents: Analytical grade reagents such as inorganic salts and buffer solutions required for the experiment are all supplied by China National Pharmaceutical Group Co., Ltd.

[0048] 3. Microbial culture system (1) LB medium components: This medium contains the following components (g / L): tryptone 10.0, yeast extract 5.0, sodium chloride 10.0. It is mainly used for the recovery of strains and the proliferation of plasmids. The pH value is adjusted to 7.0-7.2 with NaOH solution.

[0049] (2) TB medium formulation: tryptone 12 g / L, yeast extract 24 g / L, glycerol 5 g / L, KH2PO4 2.31 g / L, K2HPO4·3H2O 16.43 g / L. The high-density fermentation medium consists of the following components (g / L): tryptone 12.0, yeast extract 24.0, glycerol 5.0 (as carbon source), potassium dihydrogen phosphate (KH2PO4) 2.31, and dipotassium hydrogen phosphate trihydrate (K2HPO4·3H2O) 16.43. This system maintains the pH within the range of 6.8-7.0 through a phosphate buffer system, making it suitable for the induction and expression of recombinant strains.

[0050] 4. Methods for analyzing the catalytic activity of keratinase (1) Substrate system construction: Accurately weigh 0.1046 g of the sample. p NPB was diluted to volume in a 10 mL volumetric flask with acetonitrile to prepare a 50 mmol / L stock solution, which was then sealed and stored at -20°C. This small molecule ester compound serves as a standard model substrate for keratinase, and the butyrate ester bond in its molecule can characterize the intrinsic hydrolytic ability of the enzyme's catalytic center.

[0051] (2) Enzyme activity detection procedure: First, in the pre-equilibration stage of the reaction system, the 10 mmol / L Tris-HCl buffer (pH 8.0) was preheated to 37°C, and 1.44 mL was transferred to a 0.5 cm optical path quartz cuvette. Next, in the enzyme concentration optimization stage, the enzyme sample was serially diluted with buffer to ensure that the absorbance change at 405 nm was within the linear range of 0.2-0.8 during the initial reaction rate measurement. Finally, in the reaction initiation and monitoring stage, 30 μL of diluted enzyme solution was injected sequentially (vortexed for 5 seconds) and 30 μL of... p The NPB stock solution was immediately placed in the sample chamber of a visible spectrophotometer, and the absorbance kinetic curve at a wavelength of 405 nm was continuously recorded over 60 seconds under a constant temperature of 37°C.

[0052] (3) Detection principle and unit definition: p NPB releases p-nitrophenol after enzymatic hydrolysis. p -nitrophenol, p The product (NP) ionizes in an alkaline environment to form a yellow quinone structure, producing a characteristic absorption peak at 405 nm.

[0053] Enzyme activity unit (U) is defined as: the amount of enzyme that can be catalyzed to produce 1 μmol per minute under standard detection conditions (37℃, pH 8.0). p The amount of enzyme required for NP (4) Industrial relevance verification: choose p NPB serves a dual purpose as a characterizing substrate: firstly, it represents the catalytic mechanism; secondly, it is representative of the catalytic mechanism. p Both NPB and PVAc contain crucial ester bonds. The core catalytic function of keratinase is the hydrolysis of these ester bonds. Therefore, through... p The enzymatic properties measured using NPB as a small molecule model substrate can reflect the intrinsic activity of the keratinase catalytic center. Secondly, industrial suitability is crucial; the enzyme activity was measured at pH 8.0 and 60°C, conditions suitable for the white water circulation system in the pulp and paper industry. Mutants that yield excellent data at pH 8.0 and 60°C indicate superior performance in the pulp and paper industrial environment.

[0054] 5. Evaluation of the hydrolysis efficiency of polyvinyl acetate substrates (1) Substrate system construction: Dissolve PVAc powder in dimethyl sulfoxide (DMSO) solvent to prepare a stock solution with a concentration of 2% (w / v).

[0055] (2) Enzymatic hydrolysis reaction process: The hydrolysis system was constructed in a 50 mM Tris-HCl buffer (pH 8.0) reaction system according to the following sequence: First, the corresponding volume of buffer solution was added to a screw-cap glass vial for preheating at a specified temperature; second, 10 μg of purified keratinase protein to be tested was added; subsequently, 1.0 mL of PVAc stock solution was transferred to the vial and mixed thoroughly; finally, the vial was placed in a 60°C water bath with a shaker and the reaction was continued at 150 rpm for 12 hours. A sealed device was used throughout the reaction to prevent concentration deviations caused by solvent evaporation.

[0056] (3) Quantitative analysis of acetic acid release: After the reaction, the supernatant was collected and the released acetic acid was quantitatively analyzed spectrophotometrically using an acetic acid assay kit. The specific order of addition of the additives was as shown in Table 1. Sample measurements were performed, and single-point or curve calibration was used to detect the change in absorbance at 340 nm (A2-A1). The results were then used to calculate the acetic acid content by substituting the values ​​into the standard curve.

[0057] Table 1. Order of addition for acetic acid concentration determination

[0058] 6. Determination of the enzymatic properties of keratinase M8 and its mutants: (1) Optimal temperature: In a pH 8.0 Tris-HCl buffer system, a temperature gradient response model was established by modifying the measurement temperature according to the method described in "4. Analysis Method of Keratinase Catalytic Activity". A test range of 40°C to 90°C (10°C intervals) was set, and the catalytic activity of M8 and its mutants was measured. Using the highest activity value as a baseline (100%), the relative activity values ​​at each temperature point were calculated, and a temperature-enzyme activity response curve was constructed to determine the optimal operating temperature.

[0059] (2) Thermal dissipation dynamics study: To determine the heat tolerance of the enzyme molecule, the enzyme solution was incubated in a 70°C constant temperature water bath using a gradient method to determine the half-life (t) of M8 and its mutants. 1 / 2 After sampling at set times, the reaction was immediately terminated by ice bath, and the remaining keratinase activity was measured according to the method described in "4. Keratinase Catalytic Activity Analysis". The first-order rate constant was obtained by linear regression of ln (remaining activity percentage) with incubation time. k d The formula used is: lnA = k d ×t (A: percentage of remaining activity, t: incubation time). Then... k d Calculate its half-life at different temperatures. The formula for calculating the half-life is t. 1 / 2 = ln2 / kd. This parameter characterizes the operational stability of the enzyme under industrial high-temperature environments and is used to calculate its half-life at 70°C.

[0060] (3) Optimal pH: Following the method described in "4. Keratinase Catalytic Activity Analysis Method" above, the hydrolytic activity of keratinase was determined by modifying the pH value. Three buffer systems were used to cover the pH range of 6.0–11.0: phosphate buffer (pH 6.0–8.0, 100 mM), Tris-HCl buffer (pH 8.0–9.0, 100 mM), and sodium carbonate buffer (pH 9.0–11.0, 100 mM). The hydrolytic activity was determined under constant temperature conditions of 37°C by… p The NPB hydrolysis rate was determined by measuring enzyme activity at various pH values. Using peak activity as a reference (100%), a pH-activity distribution map was plotted to identify the optimal pH and effective catalytic range.

[0061] (4) pH stability: The enzyme preparations were pre-incubated in the following buffer systems (37°C, 24 h), and the residual activity of the keratinase was determined according to the method described in "4. Analysis Method for Keratinase Catalytic Activity". Four buffer systems were used to cover the pH range of 6.0–11.0: citrate buffer (pH 3.0–6.0, 100 mM), phosphate buffer (pH 6.0–8.0, 100 mM), Tris-HCl buffer (pH 8.0–9.0, 100 mM), and sodium carbonate buffer (pH 9.0–11.0, 100 mM). After incubation, the residual activity was measured (initial activity set at 100%), and pH-stability correlation curves were constructed to evaluate the enzyme's conformational retention ability under different pH conditions.

[0062] 7. The molecular biology operations involved in this patent, including but not limited to plasmid construction, protein purification, and nucleic acid analysis, all follow the classic methods established by J. Sambrook in *Molecular Cloning: A Laboratory Manual* (3rd edition). Operations involving commercially available kits are strictly performed according to the technical specifications provided by the manufacturer, and key parameters, including reaction temperature, duration, and sample loading volume, have been optimized through pre-experimental verification.

[0063] Example 1: Preparation of recombinant keratinase and its mutants Keratinase M8 is a mutation of the wild-type keratinase. Its performance in degrading PVAc at lower temperatures (50°C) is comparable to that of the wild type, but at 60°C, it is twice as effective as the wild type.

[0064] The gene encoding keratinase M8 (nucleotide sequence shown in SEQ ID NO.5) was ligated to the multiple cloning restriction site of the pET20b(+) plasmid to obtain the recombinant plasmid pET20b(+). -m8 The recombinant plasmid was then transformed into Escherichia coli JM109 to obtain a recombinant Escherichia coli strain. JM109 / m8 The above recombinant plasmid was transformed into E. coli BL21(DE3) pLysS ， Obtain recombinant Escherichia coli strain E. coli BL21(DE3) pLysS / pET20b(+)- m8 .

[0065] Mutants with enhanced PVAc degradation performance were identified using methods such as structural alignment, virtual screening, and Funclib-assisted prediction. The pET-20b(+)- m8 Using the parental template, site-directed mutagenesis was performed using whole-plasmid PCR. Mutants containing single amino acid substitutions were constructed using 2×PhantaMasterMix and mutation primers. Restriction enzymes were used to eliminate the parental template. DpnThe PCR products were then processed using the first step. Subsequently, they were transformed into *E. coli* JM109 competent cells and screened on LB agar plates supplemented with ampicillin (100 μg / mL). Positive colonies were isolated and sequenced using bidirectional Sanger sequencing.

[0066] Recombinant plasmids carrying the keratinase gene and its mutants were transformed into *E. coli* BL21(DE3) pLysS cells and screened on antibiotic-containing plates. All culture media contained ampicillin (100 μg / mL). Single colonies were cultured in 10 mL of LB medium at 37°C and 220 rpm / min for 12 hours. To optimize the growth of the strain and the expression of the target protein, activated strain cells were inoculated at a 1% inoculum into 100 mL of TB medium and incubated at 37°C and 220 rpm / min for 2–3 hours to achieve the desired cell density (OD). 600 When the saturation reached 0.7-0.8, the temperature was adjusted to 25℃, and fermentation continued for 48 hours. After culturing, the cultured cells were centrifuged at 12000 g for 20 minutes, and the supernatant containing crude enzyme was collected for further analysis. Subsequently, the keratinase and its mutant were purified using a Ni-NTA column. First, the purification column was equilibrated with buffer A (25 mM Tris, 500 mM NaCl, pH 7.4). The fermentation supernatant was filtered through a 0.22 μm membrane and loaded onto the column. Then, the protein was eluted with a gradient of buffer A and buffer B (25 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.4). The eluted protein was collected and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After purification by the nickel column, the SDS-PAGE results showed that the recombinant keratinase and its mutant were expressed in E. coli. The results showed that the protein reached electrophoretic purity, and the size of the purified protein was approximately 20.25 kDa. Figures 1-2 ).

[0067] Example 2: Comparison of the performance of recombinant keratinase and its single-point mutant in degrading PVAc Polyvinyl acetate (PVAc) is a common pressure-sensitive adhesive in papermaking and a major component causing adhesive deposition problems. Keratinase can hydrolyze the ester bonds of PVAc, breaking it down into acetic acid and polyvinyl alcohol, thereby reducing its viscosity. Typically, the processing temperature for waste paper pulp is maintained at around 60°C, but equipment aging or environmental fluctuations may cause the temperature to exceed this threshold. To evaluate its applicability under harsh conditions, this study investigated the degradation efficiency of PVAc by keratinase at high temperatures. The experiment was conducted in a shaker at 60°C and 150 rpm for 12 hours. The total reaction volume was 10 mL, containing: 1 mL of substrate solution (containing 2% w / v PVAc) dissolved in dimethyl sulfoxide, 10 μg of keratinase, and 50 mM Tris-HCl buffer (pH 8.0). After the reaction, the acetic acid released by the enzyme was quantitatively analyzed spectrophotometrically using a Megazyme acetic acid assay kit.

[0068] like Figure 3 , Figure 4 and Figure 5 As shown, the performance of mutants designed using three different strategies (structure alignment, virtual screening, and Funclib prediction) in degrading PVAc was investigated. Figure 3 The present invention screens mutants that improve the degradation performance of PVAc using a structure comparison strategy. The results show that the L174F mutant can increase the acetic acid production, with an acetic acid production of 14.17 mg / L, which is 1.24 times higher than that of M8. Figure 4 This invention uses a virtual screening strategy to screen mutants that improve PVAc degradation performance. The results show that mutants T29A, T29L, T37V and L66W can increase acetic acid production, with acetic acid production of 12.39 mg / L, 12.93 mg / L, 11.85 mg / L and 12.18 mg / L, respectively, which are 1.08, 1.13, 1.03 and 1.06 times higher than M8. Figure 5 This invention uses a Funclib-assisted prediction strategy to screen mutants that enhance PVAc degradation performance. Results show that mutants T29G, T37S, L66P, ​​I168V, and L174W can increase acetic acid production, with yields of 13.04 mg / L, 12.29 mg / L, 12.73 mg / L, 13.18 mg / L, and 14.22 mg / L, respectively, representing increases of 1.14, 1.07, 1.11, 1.15, and 1.24 times compared to M8. In conclusion, mutants T29G, T29A, T29L, T37S, T37V, L66P, ​​L66W, I168V, L174F, or L174W can enhance PVAc degradation performance.

[0069] Example 3: Comparison of the performance of recombinant keratinase and its combined mutants in degrading PVAc To further improve the performance of PVAc degradation, iterative combinations of single-point mutants were performed. First, the mutants M8 / L174F(C9) and M8 / L174W, which have good degradation performance, were used as starting mutants to evaluate their performance in degrading PVAc.

[0070] Figure 6 This invention screened combined mutants with improved PVAc degradation performance using M8 / L174F as a template. The results showed that the combined mutants M8 / L174F / T29G, M8 / L174F / T29A, M8 / L174F / T29L, M8 / L174F / T37S, M8 / L174F / L66P, ​​and M8 / L174F / I168V showed increased acetic acid production compared to M8 / L174F. Their acetic acid production was 16.15 mg, 14.60 mg, 15.45 mg, 14.31 mg, 16.44 mg, and 17.57 mg, respectively, which were 1.41, 1.27, 1.35, 1.25, 1.43, and 1.53 times higher than that of M8.

[0071] Figure 7 This invention uses M8 / L174W as a template to screen for combined mutants with improved PVAc degradation performance. Results show that the combined mutants M8 / L174W / T29G, M8 / L174W / L66P, ​​and M8 / L174W / L66W exhibited increased acetic acid production compared to M8 / L174W, with yields of 15.50 mg, 15.93 mg, and 16.07 mg, respectively, representing increases of 1.35, 1.39, and 1.40 times compared to M8. In conclusion, the combined mutant M8 / L174F / I168V demonstrates the best PVAc degradation performance and will therefore be used as the template for the next round of iterative mutations.

[0072] like Figure 8 As shown, this invention uses M8 / L174F / I168V (C10) as a template to screen for combined mutants with improved PVAc degradation performance. The results show that the combined mutants M8 / L174F / I168V / T29G, M8 / L174F / I168V / T29L, and M8 / L174F / I168V / L66P exhibited increased acetic acid production compared to M8 / L174F / I168V, with yields of 19.85 mg, 17.92 mg, and 17.92 mg, respectively, representing increases of 1.73, 1.56, and 1.56 times compared to M8.

[0073] Figure 9In this invention, M8 / L174F / I168V / T29G was used as a template to screen for combined mutants that improved the degradation performance of PVAc. Further superposition showed that the acetic acid production was not increased. Therefore, the optimal mutant was M8 / L174F / I168V / T29G (C11).

[0074] Example 4: Determination of the half-life of recombinant keratinase and its combined mutants The ability of an enzyme to retain its activity after undergoing irreversible denaturation is called kinetic stability. Parameter t 1 / 2 This is the time required for enzyme activity to decrease to half of its initial value, and is commonly used to evaluate kinetic stability. p Using NPB as a substrate, the half-life of keratinase M8 and its iterative mutant combinations (C9, C10, and C11) at 70°C was systematically analyzed. The specific steps were as follows: appropriately diluted protein samples were incubated in a 70°C water bath at different time intervals, and the remaining enzyme activity of the keratinase was measured according to the method described in "4. Keratinase Activity Assay" above. The first-order rate constant was obtained by linear regression of ln (remaining activity percentage) with incubation time. k d The formula used is: lnA = k d ×t (A: percentage of remaining activity, t: incubation time). Then... k d Calculate its half-life at different temperatures. The formula for calculating the half-life is t. 1 / 2 = ln2 / k d .

[0075] Figure 10 The half-life t of M8 and its series of combined mutants at 70°C is given by the present invention. 1 / 2 Compared to keratinase M8, the engineered mutant combinations C9, C10, and C11 exhibited improved half-lives at 70°C. The half-lives of mutants M8, C9, C10, and C11 at 70°C were 39.8 h, 66.7 h, 78.9 h, and 205.9 h, respectively. Among them, mutant C11, exhibiting the best PVAc degradation performance, showed a 5.17-fold increase in half-life compared to M8 at 70°C, further broadening its industrial application potential in high-temperature environments.

[0076] Example 5: Optimal temperature of recombinant keratinase and its combined mutants To investigate the optimal temperature for wild-type and combinatorial mutant keratinase, the measurement temperature was adjusted according to the method described in "4. Keratinase Activity Assay" above. p Using NPB as a substrate, the enzyme activity of the recombinant enzyme was measured at 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃. Figure 11 The figure shows the optimal temperature for M8 and its series of combined mutants of the present invention. The results show that the optimal temperatures of the engineered combined mutants C9, C10, and C11 are consistent with those of M8, with an optimal temperature of 90°C or higher. This characteristic is largely attributed to the high thermal stability of the mutants, which greatly expands the application range of these mutants.

[0077] Example 6: Optimal pH and pH stability of recombinant keratinase and its combined mutants To investigate the optimal pH for wild-type and combinatorial mutant keratinase, the hydrolytic activity of keratinase was measured by changing the pH of the buffer solution according to the method in Example 4. The buffer solutions were as follows: phosphate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 8.0-9.0), and sodium carbonate buffer (pH 9.0-11.0). The hydrolytic activity of keratinase at pH 6.0-10 was measured at 37°C. p NPB hydrolysis activity. Figure 12 The optimal pH for mutant M8 and its series of combined mutants was determined. Results showed that the optimal pH for mutant M8 was 9.0. With the accumulation of mutations, the optimal pH for mutant C11 changed to 9.5, indicating improved performance under alkaline conditions. This demonstrates that the thermal stability and optimal pH of mutant C11 were optimized.

[0078] In addition, to investigate the pH stability of wild-type and combinatorial mutants of keratinase, keratinase M8 and its mutants were diluted to a certain extent with buffers of different pH values, stored at 37°C in different buffers for 24 hours, and the residual activity of keratinase was measured according to the method in Example 4. The buffers are as follows: citrate buffer (pH 3.0~6.0), phosphate buffer (pH 6.0~8.0), Tris-HCl buffer (pH 8.0~9.0), and sodium carbonate buffer (pH 9.0~11.0). The pH stability of M8 and its series of combinatorial mutants is as follows. Figure 13 As shown in the figure, both M8 and the mutant maintained over 80% of their catalytic activity within a pH range of 4.0–10.0. Above pH 10, the stability of M8 decreased slightly, maintaining 76.85% and 68.15% of its activity at pH 10.5 and 11.0, respectively. In contrast, with the accumulation of iterative mutations, the mutant exhibited superior pH stability compared to M8. The mutant C11 maintained 91.55% and 88.38% of its activity at pH 10.5 and 11, respectively, representing increases of 14.70% and 20.23% compared to M8. Overall, the enhanced stability of mutant C11 in alkaline environments indicates its higher alkali tolerance, broadening its application potential under alkaline conditions.

[0079] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A keratinase mutant for degrading PVAc, characterized in that, The mutation is made at one or more residue sites at 29threonine, 37threonine, 66thleonine, 168th isoleucine, 169th isoleucine, and 174thleonine, relative to the keratinase parental amino acid sequence shown in SEQ ID NO.

1.

2. The keratinase mutant according to claim 1, characterized in that, It has any of the mutations (a) to (h); (a) Mutate the threonine at position 29 of the amino acid sequence shown in SEQ ID NO.1 to glycine, alanine, or leucine; (b) Mutate the threonine at position 37 of the amino acid sequence shown in SEQ ID NO.1 to serine or valine; (c) Mutate leucine at position 66 of the amino acid sequence shown in SEQ ID NO.1 to proline or tryptophan; (d) Mutate isoleucine at position 168 of the amino acid sequence shown in SEQ ID NO.1 to valine; (e) Mutate leucine at position 174 of the amino acid sequence shown in SEQ ID NO.1 to phenylalanine or tryptophan; (f) Mutate leucine at position 174 of the amino acid sequence shown in SEQ ID NO.1 to phenylalanine and isoleucine at position 168 to valine; (g) Mutate leucine at position 174 of the amino acid sequence shown in SEQ ID NO.1 to tryptophan, and leucine at position 66 to tryptophan; (h) The amino acid sequence shown in SEQ ID NO.1 is mutated by changing leucine at position 174 to phenylalanine, isoleucine at position 168 to valine, and threonine at position 29 to glycine.

3. The gene encoding the mutant of claim 1 or 2.

4. A recombinant vector comprising the gene of claim 3.

5. The recombinant vector according to claim 4, characterized in that, Choose pET series or pPIC9K expression vectors.

6. A recombinant microorganism expressing the mutant of claim 1 or 2.

7. A method for preparing the keratinase mutant according to claim 1 or 2, characterized in that, The keratinase mutant is expressed using the recombinant vector of claim 4 or the recombinant microorganism of claim 6.

8. A method for improving the catalytic performance of keratinase, characterized in that, Mutate the keratinase with the amino acid sequence shown in SEQ ID NO.1; the mutation is selected from single mutants T29G, T29A, T29L, T37S, T37V, L66P, ​​L66W, I168V, L174F, or L174W; or, Combinatorial mutants: L174F / I168V, L174W / L66W, L174F / I168V / T29G.

9. The application of the keratinase mutant according to claim 1 or 2.

10. The application according to claim 9, characterized in that, The applications include, but are not limited to, polyester degradation and detergent enhancement.

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