Collagenase mutant as well as preparation method and application thereof

Abstract

The invention discloses a collagenase mutant as well as a preparation method and application thereof. Compared with the amino acid sequence of wild type collagenase, the collagenase mutant has the advantages that the 23rd tryptophan is mutated into histidine, the 26th isoleucine is mutated into glutamine, the 289th glutamic acid is mutated into histidine, and the 486th tryptophan is mutated into alanine. The collagenase mutant provided by the invention can effectively degrade collagen, and has important practical significance on application of collagenase and effective utilization of collagen resources.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and protein engineering technology, specifically to a collagenase mutant, its preparation method, and its application. Background Technology

[0002] Collagen is a biological macromolecule protein and a major component of the extracellular matrix. It not only provides cells with a flexible scaffold but also regulates vital cellular processes, including differentiation, cell growth, survival, and migration. It is also one of the key materials in the biotechnology industry and has wide applications in medicine, food, and daily chemicals.

[0003] Collagen has a complex molecular structure and good stability, making it difficult to be degraded by common proteases. Matrix metalloproteinases (MMPs) and microbial collagenases are among the few known enzymes that can degrade collagen in situ in physiological environments, and can maintain high enzyme activity in such environments to perform their degradation function.

[0004] Microbial collagenases have advantages such as being secreted extracellularly, easy to isolate and extract, larger molecular weight, more complex structural domains, stronger substrate adaptability, and the ability to generate enzyme activity without activating zymogens. Among them, Col H collagenase, Col G collagenase, and Col A collagenase from Clostridium perfringens are called "true" collagenases because they can cleave triple-helix collagen; in addition, Col A collagenase extracted from Bacillus cereus has also been added to the collection of "true" collagenases.

[0005] Currently, commercially available collagenases are mainly extracted from Clostridium histolytica. Although they are recognized as the standard enzyme for identifying novel collagenases, their high preparation cost and the pathogenicity of certain strains limit their application safety and large-scale production. Bacillus cereus and its closely related family members are ubiquitous Gram-positive, sporulating facultative anaerobic bacteria in the environment. However, as opportunistic pathogens, they have become a cause of foodborne gastrointestinal and non-gastrointestinal diseases, and their direct application also poses safety risks.

[0006] Given the above problems, it is of great significance to construct a collagenase-producing strain with high expression and no pathogenicity; currently, the expression level of collagenase in engineered strains is limited, which restricts its practicality. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a collagenase mutant, its preparation method, and its applications. This invention optimizes the performance of ColA collagenase from two key dimensions by mutating its amino acid sequence: increasing soluble expression levels and enhancing its enzymatic activity in hydrolyzing collagen. This achieves highly efficient soluble expression and high enzyme activity of ColA, providing a solution for safe and low-cost collagenase production.

[0008] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a collagenase mutant, wherein, compared with the amino acid sequence of wild-type collagenase, tryptophan at position 23 is mutated to histidine, isoleucine at position 26 is mutated to glutamine, glutamic acid at position 289 is mutated to histidine, and tryptophan at position 486 is mutated to alanine; the amino acid sequence of the wild-type collagenase is shown in SEQ ID NO: 1.

[0009] Furthermore, the amino acid sequence of the collagenase mutant is shown in SEQ ID NO: 2.

[0010] The present invention also provides a gene encoding the collagenase mutant described above, the nucleotide sequence of which is shown in SEQ ID NO: 4.

[0011] The present invention also provides a method for preparing the collagenase mutant, comprising the following steps: (1) Obtain the gene sequence of the collagenase mutant, add Ndel and HindIII restriction sites to both ends of the gene, and double digest the gene with Ndel and HindIII to obtain the collagenase mutant gene fragment. (2) The vector pET30a was digested with Ndel and HindIII to obtain the vector fragment; (3) The collagenase mutant gene fragment and the vector fragment are ligated to obtain a collagenase recombinant expression vector; (4) The collagenase recombinant expression vector was transferred into the host bacterium Escherichia coli, and the protein was expressed by shaking culture. The bacteria were broken to obtain crude enzyme solution, and the crude enzyme solution was purified to obtain collagenase mutant.

[0012] Furthermore, the ligase used for ligation is T4 ligase, and the host bacterium is Escherichia coli.

[0013] The present invention also provides a collagenase recombinant expression vector, wherein the vector includes the aforementioned gene and the expression vector is pET30a.

[0014] The present invention also provides a collagenase expression strain, the strain comprising the aforementioned vector, wherein the host bacterium of the strain is Escherichia coli BL21.

[0015] The present invention also provides the application of any one of the following in catalytic collagen hydrolysis: 1) the collagenase mutant described above; 2) The aforementioned carrier; 3) The strain described above.

[0016] The present invention also provides an application of the collagenase mutant in the production of glycyl-prolyl-alanine. Glycyl-prolyl-alanine can be generated by mixing fish skin gelatin solution and collagenase mutant and then incubating.

[0017] Furthermore, the concentration of the fish skin gelatin solution is 15-25 g / L; the enzyme activity of the collagenase mutant added to the fish skin gelatin solution is 1-1.1 U / ml; The incubation conditions are 30~55℃ and pH 4~9.

[0018] Furthermore, the concentration of the fish skin gelatin solution is 20 g / L; the volume ratio of the collagenase mutant to the fish skin gelatin solution is 1:6.5. The incubation conditions were 30°C and pH 7.

[0019] The beneficial effects of this invention are: 1. Currently, most commercially available ColA collagenases are derived from Clostridium perfringens and Bacillus cereus, both of which are pathogenic bacteria. This invention uses Escherichia coli as the expression strain, solving the problems of high preparation cost and high pathogenicity of the strains for ColA collagenase. This lays the foundation for the widespread application of collagenase and the effective utilization of collagen.

[0020] 2. Compared with the expression level of wild-type collagenase (25 mg / L), the collagenase mutant of the present invention has a protein expression level of 70 mg / L under the same conditions, which is 2.8 times that of wild-type collagenase, i.e., an increase of 180%.

[0021] 3. Compared with the specific activity of wild-type collagenase (270 U / mg), the soluble protease activity of the collagenase mutant of the present invention reaches 307 U / mg, which is 1.137 times that of wild-type collagenase, that is, the specific activity of the enzyme is increased by 13.7%. Attached Figure Description

[0022] Figure 1 A comparison of the molecular dynamics simulation characteristics of wild-type collagenase (WT) and collagenase mutant (MUT); Figure 2 This is a schematic diagram of the key residue (Trp23→His23) of the collagenase mutant (MUT) and its surrounding molecular interactions. Figure 3 This is a schematic diagram of the key residue (Ile26→Gln26) of the collagenase mutant (MUT) and its surrounding molecular interactions. Figure 4 This is a schematic diagram of the key residues (Glu289→His289) of the collagenase mutant (MUT) and their interactions with surrounding molecules. Figure 5 This is a schematic diagram of the key residues (Trp486→Ala486) of the collagenase mutant (MUT) and their interactions with surrounding molecules. Figure 6 This is a diagram illustrating the construction of the mutant plasmid in an embodiment of the present invention; Figure 7 SDS-PAGE analysis of the collagenase mutant cloned in pET30a and expressed in BL21(DE3) strain; In the figure, M: Protein marker; PC1: 1 μg BSA; PC2: 2 μg BSA; NC: Uninduced cell lysate; NC1: Uninduced cell lysate supernatant; NC2: Uninduced cell lysate precipitate; 1: Cell lysate induced by 0.5 mM IPTG at 15°C for 16 hours; 2: Cell lysates induced by 0.1 mM IPTG at 30°C for 3 hours; 3: Cell lysate supernatant after induction with 0.5 mM IPTG at 15℃ for 16 hours; 4: Cell lysate supernatant after induction with 0.1 mM IPTG at 30℃ for 3 hours; 5: Cell lysate precipitate after 16 hours of induction with 0.5 mM IPTG at 15°C; 6: Cell lysate precipitate after induction with 0.1 mM IPTG at 30℃ for 3 hours; Figure 8 SDS-PAGE analysis of wild-type collagenase cloned in pET30a and expressed in BL21(DE3) strain; In the diagram, M: Protein marker; 1:1 μg wild-type collagenase ColA; 2:2 μg wild-type collagenase ColA; 3: Cell lysate supernatant after induction with 0.5 mM IPTG at 15℃ for 16 hours; 4: Cell lysate precipitate after 16 hours of induction with 0.5 mM IPTG at 15℃; Figure 9The graph shows the effect of pH on the collagenase mutant (MUT). Figure 10 The graph shows the effect of temperature on the collagenase mutant (MUT). Figure 11 This is a standard curve of GPA tripeptide. Figure 12 This is a comparison chart of GPA content in the enzymatic hydrolysis products. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0024] Experimental materials 1. LB solid medium 1000mL: tryptone 10g, yeast extract 5g, NaCl 10g, agar powder 16g; pH 7.0, sterilize at 121℃ for 20min.

[0025] 2. LB liquid culture medium 1000mL: tryptone 10g, yeast extract 5g, NaCl 10g; pH 7.0, sterilize by moist heat at 121℃ for 20min.

[0026] 3. Equilibration buffer: 20mM PBS, 0.5M NaCl, pH 7.4.

[0027] 4. Washing buffer: 20mM PBS, 0.5M NaCl, 0~30mM imidazole, pH 7.4.

[0028] 5. Elution buffer: 20mM PBS, 0.5M NaCl, 50~500mM imidazole, pH 7.4.

[0029] Example 1 Screening and construction of collagenase mutant mutation sites 1. The amino acid sequence of wild-type collagenase ColA is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 3. A literature review of the ColA collagenase domains revealed that the central domain for hydrolyzing collagen is the AD domain. Through the continuous strengthening of the binding to collagen via the CBD and PKD domains, the enzyme conformation changes from a "closed state" to a "semi-closed-semi-relaxed state," completing the unwinding of collagen and exposing its polypeptide single chains for hydrolysis by the AD domain. Therefore, the domains in which wild-type collagenase ColA catalyzes its activity are Try93-Lys960.

[0030] The protein sequence was optimized using the AI-DeZyme algorithm, and specific amino acid sites were mutated in the collagenase mutant ColA-W23H / I26Q / E289H / W486A.

[0031] 2. Compared to the amino acid sequence of wild-type collagenase ColA, the collagenase mutant ColA-W23H / I26Q / E289H / W486A, based on the amino acid sequence shown in SEQ ID NO: 1, mutated tryptophan at position 23 to histidine, isoleucine at position 26 to glutamine, glutamic acid at position 289 to histidine, and tryptophan at position 486 to alanine, finally yielding the amino acid sequence of collagenase mutant ColA-W23H / I26Q / E289H / W486A as shown in SEQ ID NO: 2, and its nucleotide sequence as shown in SEQ ID NO: 4.

[0032] 3. Molecular dynamics simulations were performed on wild-type collagenase ColA (WT) and collagenase mutant ColA-W23H / I26Q / E289H / W486A (MUT), and the results were analyzed using four core indicators: root mean square fluctuation of residues (RMSF), solvent accessible surface area (SASA), radius of gyration (Rg), and root mean square deviation (RMSD).

[0033] The results are as follows Figure 1 As shown, the collagenase mutant ColA-W23H / I26Q / E289H / W486A has three major advantages: First, the RMSF is reduced, which inhibits excessive conformational fluctuations of the protein, improves conformational stability, and avoids the collapse of the active site; second, the SASA is increased, which increases the solvent exposure of the active pocket and reduces the substrate binding barrier; third, the Rg is slightly lower and the RMSD is more stable in the later stage of simulation, achieving a dynamic balance of "global compactness-local openness" to ensure substrate recognition efficiency.

[0034] 5. Analysis was performed on four key residue mutation sites: Trp23→His23, Ile26→Gln26, Glu289→His289, and Trp486→Ala486.

[0035] The results are as follows Figures 2-5As shown, the mechanisms of action of residue mutation sites vary but all contribute to enhancing enzyme activity. Trp23→His23 simplifies the hydrogen bond network, reduces steric hindrance by utilizing His's smaller side chain to improve the openness of the active pocket, and leverages His's proton donor / acceptor properties to assist catalysis. Ile26→Gln26 replaces the original hydrophobic interactions with a newly formed hydrogen bond network, enhancing local structural stability while alleviating regional confinement and optimizing substrate binding conditions. Glu289→His289 reduces excessive hydrogen bond constraints, increases local steric openness by relying on His's shorter side chain, and His's acid-base dual properties can directly improve catalytic efficiency. Trp486→Ala486 eliminates the original hydrophobic stacking effect; Ala's small side chain with only one methyl group releases more space, while the newly constructed hydrogen bond network compensates for the loss of stability, effectively enhancing the flexibility and accessibility of the active pocket.

[0036] In summary, the collagenase mutant ColA-W23H / I26Q / E289H / W486A enhances enzyme activity through a "stability-flexibility synergistic optimization": weakening excessive hydrophobic constraints or hydrogen bond networks, releasing the local flexibility and spatial openness of the active site; simultaneously, by adding hydrogen bonds or introducing residues with specific chemical functions (such as His), it maintains the overall structural stability of the protein and the necessary catalytic functional site characteristics. This mechanism allows the active site to maintain global stability while facilitating substrate entry, binding, and catalytic reactions, providing a clear molecular basis and design justification for the performance optimization of collagenase mutants.

[0037] Example 2 Constructing the collagenase mutant strain ColA-W23H / I26Q / E289H / W486A 1. Gene synthesis and subcloning Combination Figure 6 As shown, the target gene of the collagenase mutant ColA-W23H / I26Q / E289H / W486A was synthesized through whole-gene synthesis. Ndel and HindIII restriction sites were added to both ends of the gene, and the target gene was double-digested with Ndel and HindIII. The gene was then ligated with the pET30a vector, which had also been digested with Ndel and HindIII, using T4 ligase. The ligation was carried out overnight at 16°C to obtain the collagenase recombinant expression vector pET30a-ColA-W23H / I26Q / E289H / W486A.

[0038] 2. Recombinant plasmid-transformed expression strains (1) Take out Escherichia coli BL21(DE3) competent cells from the -80℃ freezer and place them on ice to thaw slowly.

[0039] (2) Add 100 ng of collagenase recombinant expression vector pET30a-ColA-W23H / I26Q / E289H / W486A to competent cells and mix gently.

[0040] (3) Incubate the mixture in an ice bath for 30 minutes.

[0041] (4) Perform heat shock treatment: Place the mixture in a 42°C water bath and let it stand for 90 seconds, avoiding shaking during the process.

[0042] (5) After heat shock, quickly transfer to an ice bath and let stand for 3 minutes.

[0043] (6) Add 100 μL of room temperature LB liquid medium to the mixture.

[0044] (7) Place the cells in a shaker at 37°C and shake at 200 rpm for 60 minutes to allow the cells to recover and express the resistance gene.

[0045] (8) Spread the above bacterial solution onto an LB solid agar plate containing 50 μg / mL kanamycin.

[0046] (9) Invert the plate and place it in a 37°C incubator overnight.

[0047] After verification, the collagenase mutant ColA-W23H / I26Q / E289H / W486A expression strain ColA-MUT was obtained.

[0048] Through the above steps, the wild-type collagenase ColA expression strain ColA-WT was obtained.

[0049] Example 3 Collagenase-induced expression and purification 1. Induced expression of ColA-MUT and SDS-PAGE analysis (1) Inoculation and culture: Three well-isolated single colonies of ColA-MUT were picked and inoculated into 4 mL LB medium containing 50 μg / mL kanamycin. The bacterial culture was placed in a shaker at 37℃ and cultured at 200 rpm until OD. 600 =0.6-0.8.

[0050] (2) Induction of expression: The cultures were divided into 3 groups and treated as follows: Experimental group 1: IPTG was added to a final concentration of 0.5 mM and induced at 15℃ for 16 h; Experimental group 2: IPTG was added to a final concentration of 0.1 mM and induced at 30℃ for 3 h; Control group: No IPTG added, cultured under the same conditions as a negative control.

[0051] (3) Expression detection: Collect bacterial cells from each group and analyze the expression level and solubility of the target protein by SDS-PAGE.

[0052] (4) Cell collection and lysis: Take 450 μl of culture, centrifuge to collect cell pellet; add 300 μl of lysis buffer (containing 50 mM Tris-HCl, 500 mM NaCl, 5% glycerol, 0.5% Triton X-100, pH 8.0), and sonicate for 1 minute.

[0053] (5) Preparation of cell lysis buffer sample: Take 100 μl of cell lysis buffer from step (4) and mix it with 50 μl of 5× loading buffer to obtain whole cell lysis buffer sample. Heat the sample at 100℃ for 10 minutes, then centrifuge at 15,000 rpm for 2 minutes, and wait for loading.

[0054] (6) Preparation of cell lysate supernatant and precipitate: Take the remaining 200 μl of cell lysate from step (4), centrifuge at 15,000 rpm for 10 minutes, and separate the supernatant and precipitate.

[0055] (7) Supernatant sample: Take 180 μl of the supernatant from step (6) and add 90 μl of 5× loading buffer to mix.

[0056] (8) Precipitate the sample: Resuspend all the precipitate from step (6) with 130 μl of 5× loading buffer.

[0057] (9) Both the supernatant sample and the precipitate sample were heated at 100℃ for 10 minutes, then centrifuged at 15,000 rpm for 2 minutes, and analyzed by SDS-PAGE with BSA as a control. The results are as follows: Figure 7 As shown in Table 1, the expression level of the collagenase mutant ColA-W23H / I26Q / E289H / W486A was calculated to be 100 mg / L, the solubility was 70%, and the soluble expression level was 70 mg / L.

[0058] Table 1. Expression levels of collagenase mutants ColA-W23H / I26Q / E289H / W486A 2. Low-level induction of ColA-WT expression and SDS-PAGE analysis The same method as in step 1 was used to induce expression of strain ColA-WT.

[0059] Proteins were quantified using a pre-defined purified wild-type collagenase, ColA, and the results are as follows: Figure 8As shown in Table 2, the expression level of ColA in strain ColA-WT was calculated to be 33 mg / L, the solubility was 75%, and the soluble expression level was 25 mg / L.

[0060] Table 2 Expression levels of wild-type collagenase ColA 3. Protein purification (His tag) (1) Collect 1L of strain ColA-MUT cells induced by 0.5mM IPTG at 15℃ by centrifugation, resuspend in pre-cooled PBS, and disrupt the cells using a pressure cell disruptor at 4℃ until clear. Centrifuge at 12000r / min for 30min at 4℃ and collect the supernatant. The supernatant is the crude enzyme solution of the collagenase mutant ColA-W23H / I26Q / E289H / W486A. Filter the solution with a 0.22μm or 0.45μm filter membrane before loading to reduce impurities, improve protein purification efficiency, and prevent column clogging.

[0061] (2) For specific purification steps, refer to the instruction manual: Fill the pump pipeline with deionized water. Remove the top plug of the pre-packed column and connect it to the inlet of the chromatography equipment. Then open the bottom plug and connect it to the chromatography equipment.

[0062] (3) Rinse the column with 3-5 column volumes of deionized water to remove the storage buffer. Equilibrate the column with at least 5 column volumes of equilibration buffer. The recommended flow rate is 1-5 mL / min.

[0063] (4) Load the supernatant collected in step (1) onto the sample using a pump or syringe.

[0064] (5) Wash the column with washing buffer until the UV absorption reaches a stable baseline (generally at least 10-15 column volumes).

[0065] (6) Elution buffer can be used for one-step or gradient elution. In one-step elution, 5 column volumes of elution buffer are generally sufficient. Gradient elution can use a small gradient, such as 20 column volumes or more, to separate proteins with different binding strengths.

[0066] (7) The eluted protein sample was analyzed by SDS-PAGE. The protein with higher purity was transferred to a dialysis bag and dialyzed with dialysis fluid for 2-3 days, with the dialysis fluid changed every 4-6 hours. After complete dialysis, the protein was concentrated by embedding with sucrose at 4°C.

[0067] (8) Aliquoting and freezing: The protein concentration after dialysis and concentration was determined by BCA kit, aliquoted into small tubes, and frozen at -80℃ for later use to obtain the purified collagenase mutant ColA-W23H / I26Q / E289H / W486A.

[0068] The purified wild-type collagenase ColA was obtained using the above steps.

[0069] Example 4 Enzyme activity assay 1. Preparation of sample solution Dissolve 2g of fish skin gelatin (a 99.5% purity fish skin gel sample purchased from Guangdong Huayi Food Additives) in 100ml of ultrapure water, add 2mM of dithiothreitol, and adjust the pH to 4 to obtain the fish skin gelatin solution.

[0070] 2. Enzyme activity assay (1) Dilute the protein sample (purified collagenase mutant ColA-W23H / I26Q / E289H / W486A or crude collagenase mutant ColA-W23H / I26Q / E289H / W486A) to an appropriate concentration (2 g / L) with reaction buffer (50 mM Tris-HCl, 5 mM CaCl2 and 1 μM ZnCl2, pH = 7.5). Add 20 μL of the diluted protein sample to 480 μL of fish skin gelatin solution and digest at 37°C for 30 minutes.

[0071] (2) The reaction was terminated using a quenching buffer containing 12% (w / v) PEG 6000 and 25 mM EDTA.

[0072] (3) Mix 100 μL of reaction solution with 500 μL of ninhydrin reagent and heat at 80°C for 10 minutes. After the test tube cools, add 600 μL of H2O and mix thoroughly.

[0073] (4) Measure the absorbance of the reaction mixture at 570 nm using a spectrophotometer. One unit of collagenase activity is defined as the amount of enzyme that releases 1 μg of glycine per minute under the conditions used.

[0074] 3. The crude enzyme activities of wild-type collagenase ColA and the collagenase mutant ColA-W23H / I26Q / E289H / W486A were determined using the above methods. Enzyme activity was defined as the amount of enzyme that releases 1 μg of glycine per minute under the conditions used. Table 3 shows the crude enzyme activity of collagenase. As shown in Table 3, the enzyme activity of the crude collagenase solution of the wild type was 3.61 ± 0.36 U / mL, while the enzyme activity of the crude collagenase solution of the mutant reached 5.77 ± 0.53 U / mL. Compared with the wild type, the enzyme activity of the mutant was increased by 60%.

[0075] This result indicates that the collagenase mutant obtained through molecular modification exhibits superior catalytic activity compared to the wild type, even in the unpurified crude enzyme solution state.

[0076] 4. The specific activities of wild-type collagenase ColA and the purified collagenase mutant ColA-W23H / I26Q / E289H / W486A were determined using the above methods. The results are shown in Table 4. The specific activity of the collagenase mutant reached 307 U / mg, which is 1.14 times that of wild-type collagenase ColA, representing a 13.7% increase in specific activity.

[0077] Table 4. Collagenase specific activity Example 5 Enzymatic property analysis 1. Determination of the optimal temperature for collagenase mutant ColA-W23H / I26Q / E289H / W486A The collagenase mutant was reacted with fish skin gelatin solution in preheated water baths at temperatures of 30℃, 37℃, 40℃, 45℃, 50℃, and 55℃. A blank control was set up at each temperature, and three parallel experimental groups were formed. The absorbance was measured at 570 nm according to the method for determining collagenase activity in Example 4. The collagenase activity of the collagenase mutant was calculated, and the enzyme activity at the optimum temperature was defined as 100%.

[0078] 2. Thermal stability of collagenase mutants The collagenase mutant was incubated in water baths at temperatures of 30℃, 37℃, 40℃, 45℃, 50℃, and 55℃ for 1 hour, and then reacted with the substrate at the optimal temperature. A blank control was set up at each temperature, and three parallel experimental groups were formed. The absorbance of collagenase was measured at 570 nm according to the method for determining collagenase activity. Enzyme solution refrigerated at 4℃ was used as a 100% activity control for the reaction, and the relative residual activity of the collagenase mutant was calculated.

[0079] 3. Effect of pH on the enzyme activity of collagenase mutants The collagenase mutant was reacted with fish skin gelatin solution in buffer solutions at pH 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively. A blank control was set up for each pH condition, and three parallel experimental groups were formed. The collagenase activity of the collagenase mutant was calculated, and the enzyme activity at the optimal pH was defined as 100%.

[0080] 4. pH stability of recombinant collagenase Collagenase mutants were mixed with buffer solutions at pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, and incubated in a 30°C water bath for 1 hour. The treated enzyme solutions were then reacted with fish skin gelatin solutions at the optimal pH. A blank control and three parallel experimental groups were included for each pH condition. Absorbance was measured at 570 nm according to the collagenase activity assay method. The relative residual activity after incubation at each pH was calculated, with the enzyme activity without pH pretreatment considered as 100%.

[0081] Generally, temperature has a significant impact on collagenase activity. This invention uses fish skin gelatin as a substrate to study the optimal reaction temperature and thermal stability of collagenase within the range of 30–55°C. The results are as follows: Figure 10 As shown, the enzyme activity reaches its maximum at 30℃, and then gradually decreases with increasing temperature. When the temperature exceeds 45℃, the enzyme activity of collagenase drops sharply to below 50%.

[0082] The collagenase activity of a reaction carried out in a 4°C refrigerated solution was defined as 100%, and the thermal stability of collagenase was studied. For example... Figure 10 As shown, collagenase exhibits the highest thermal stability at 30℃. Subsequently, as the temperature increases, the thermal stability of collagenase gradually decreases. When the temperature exceeds 45℃, the thermal stability of collagenase drops below 50%.

[0083] This invention investigates the optimal reaction pH and pH stability of collagenase within a pH range of 4–9, and the results are as follows: Figure 9 As shown, collagenase exhibits the highest enzyme activity at pH 7, and its activity remains above 50% within the pH range of 7-8. Further stability studies were conducted on collagenase after incubation at different pH values ​​for 1 hour. Collagenase showed the highest pH stability at pH 8, and its activity remained above 80% within the pH range of 7-8.

[0084] Example 6 Enzymatic hydrolysis for the production of glycyl-prolyl-alanine (GPA) Collagen hydrolysates have been reported to possess various physiological activities, among which the collagen tripeptides GPH, GPA, and GHK exhibit the most prominent activities. Collagen tripeptides are small peptides containing three amino acids, obtained from the hydrolysis of collagen or gelatin. GPH can be transported across the intestinal monolayer into the plasma and stably transported within the body, facilitating absorption; it can effectively maintain collagen structure and prevent dry skin; GHK can regulate gene-mutated cells, bringing gene expression in cancer or chronic obstructive pulmonary disease patients to a healthier state, and can also promote wound healing and tissue regeneration. A novel bioactive tripeptide (Gly-Pro-Ala) has been shown to alleviate DSS-induced colitis in vivo by reducing oxidative stress and mitigating intestinal epithelial damage.

[0085] In traditional enzymatic hydrolysis processes for collagen peptides, the hydrolysis strategy centered on endopeptides is dominant. While this process can effectively degrade collagen, it is limited by the action mechanism of endopeptides, resulting in hydrolysates with a wide molecular weight distribution and high peptide complexity. Furthermore, at the target active peptide level, collagen tripeptides with specific physiological functions, such as glycyl-prolyl-histidine (GPH), glycyl-prolyl-alanine (GPA), and glycyl-histidine-lysine (GHK), are almost undetectable, thus restricting the application of collagen peptide products.

[0086] In contrast, ColA enzymes, due to their molecular properties of recognizing and cleaving specific peptide bonds in collagen, have an action site that closely matches the requirements for collagen tripeptide production, providing a key enzymatic tool for the targeted preparation of GPA and demonstrating great technological potential.

[0087] 1. Preparation of standard solutions Accurately weigh the GPA standard substance, dissolve it in 70% methanol solution, and dilute it to 100 μg / mL. Before use, prepare a series of standard control solutions with concentrations of 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, and 800 ng / mL. The GPA tripeptide standard curve is shown below. Figure 11 As shown.

[0088] 2. Enzymatic hydrolysis reaction Preparation of fish skin gelatin solution: Weigh 2 g of fish skin gelatin (food grade), slowly add it to 100 mL of ultrapure water, stir in a 50℃ water bath until completely dissolved, and obtain a fish skin gelatin solution with a final concentration of 20 g / L. Store at 4℃ in the dark for later use.

[0089] Take 260 μL of the prepared fish skin gelatin solution and add 40 μL of the corresponding reaction solution, for a total system volume of 300 μL. The experimental groups are as follows: (1) Blank control group: 40 μL PBS buffer + 260 μL fish skin gelatin solution (2) Enzymatic hydrolysis group A: 260 μL fish skin gelatin solution + wild-type collagenase ColA (0.27U); (3) Enzymatic hydrolysis group B: 260 μL fish skin gelatin solution + collagenase mutant ColA-W23H / I26Q / E289H / W486A (0.27U); Each reaction system was incubated under optimal conditions (30℃, pH 7) for 30 min. After the reaction was completed, an equal volume of methanol was added to terminate the reaction.

[0090] 3. Analysis conditions (1) Liquid chromatography conditions Separation was performed using a Waters ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 100 mm). Mobile phase A was ultrapure water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid. Isocratic elution was used with an elution time of 5 min and an A / B volume ratio of 40:60. The flow rate was 0.35 mL / min, the column temperature was 40 °C, and the injection volume was 2 μL.

[0091] (2) Mass spectrometry conditions Electrospray ionization (ESI) was used with an ion source temperature of 500℃; the ion source voltage was 5500 V in positive ion mode and -4500 V in negative ion mode. The atomizing gas (GS I) was 50 psi, the auxiliary gas (GS II) was 60 psi, the curtain gas (CUR) was 30 psi, and the collision-induced ionization (CAD) parameter was set to Medium. In triple quadrupole (Qtrap) mode, multiple reaction monitoring (MRM) scans were performed on the target ion pairs based on the optimized declustering voltage (DP) and collision energy (CE).

[0092] GPA tripeptide has the effect of alleviating intestinal epithelial damage by reducing oxidative stress in vivo, thereby alleviating DSS-induced colitis. To analyze the practical application effect of the collagenase mutant ColA-W23H / I26Q / E289H / W486A in the degradation of fish skin gelatin, this invention uses targeted quantitative detection of the GPA tripeptide concentration in the enzymatic hydrolysis products to compare the degradation effects of wild-type collagenase and the collagenase mutant on fish skin gelatin. The results are as follows... Figure 11 and Figure 12As shown, the GPA generation in group A was 5768 ng / mL, while the GPA generation in group B increased to 91744.7 ng / mL, which is 15.91 times that of group A. This demonstrates that the collagenase mutant ColA-W23H / I26Q / E289H / W486A has superior collagen-specific degradation activity compared to wild-type collagenase.

[0093] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A collagenase mutant, characterized in that: Compared with the amino acid sequence of wild-type collagenase, the collagenase mutant has the following mutations: tryptophan at position 23 is mutated to histidine, isoleucine at position 26 is mutated to glutamine, glutamate at position 289 is mutated to histidine, and tryptophan at position 486 is mutated to alanine. The amino acid sequence of the wild-type collagenase is shown in SEQ ID NO:

1.

2. The collagenase mutant according to claim 1, characterized in that: The amino acid sequence of the collagenase mutant is shown in SEQ ID NO:

2.

3. A gene encoding the collagenase mutant of claim 1, characterized in that: The nucleotide sequence of the collagenase mutant gene is shown in SEQ ID NO:

4.

4. A method for preparing the collagenase mutant according to claim 1, characterized in that: Includes the following steps: (1) Obtain the gene sequence of the collagenase mutant, add Ndel and HindIII restriction sites to both ends of the gene, and double digest the gene with Ndel and HindIII to obtain the collagenase mutant gene fragment. (2) The vector pET30a was digested with Ndel and HindIII to obtain the vector fragment; (3) The collagenase mutant gene fragment and the vector fragment are ligated to obtain a collagenase recombinant expression vector; (4) The collagenase recombinant expression vector was transferred into the host bacteria, and the protein was expressed by shaking flask culture. The bacteria were broken to obtain crude enzyme solution, and the crude enzyme solution was purified to obtain collagenase mutant.

5. The preparation method according to claim 4, characterized in that: The ligase used for ligation is T4 ligase, and the host bacterium is Escherichia coli.

6. A collagenase recombinant expression vector, characterized in that: The vector includes the gene described in claim 3, and the expression vector is pET30a.

7. A collagenase expression strain, characterized in that: The strain includes the vector described in claim 6, and the host bacterium of the strain is Escherichia coli BL21.

8. The application of any one of the following in the catalytic hydrolysis of collagen, characterized in that: 1) The collagenase mutant according to claim 1; 2) The carrier as described in claim 6; 3) The strain according to claim 7.

9. The application of the collagenase mutant according to claim 1 in the production of glycyl-prolyl-alanine, characterized in that: Glycyl-prolyl-alanine can be generated by mixing fish skin gelatin solution with collagenase mutant and then incubating the mixture.

10. The application according to claim 9, characterized in that: The concentration of the fish skin gelatin solution is 15-25 g / L; the enzyme activity of the collagenase mutant added to the fish skin gelatin solution is 1-1.1 U / ml. The incubation conditions are 30~55℃ and pH 4~9.

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