Collagenase mutant, gene fragment, recombinant plasmid, recombinant expression system, and use thereof, and methods for preparing collagenase mutant and collagen tripeptide

Abstract

Provided is a collagenase mutant KT231 that is secreted and expressed in Streptomyces lividans TK24, wherein the collagenase mutant KT231 can enzymatically hydrolyze walleye pollock skin at 60°C and can be used for preparing collagen tripeptide from walleye pollock skin.

Description

A collagenase mutant, gene fragment, recombinant plasmid, recombinant expression system and their uses, and a method for preparing collagenase mutant and collagen tripeptide. Technical Field

[0001] This invention belongs to the fields of genetic engineering and fermentation engineering, and specifically relates to a collagenase mutant, gene fragment, recombinant plasmid, recombinant expression system and its uses, as well as a method for preparing collagenase mutant and collagen tripeptide. Background Technology

[0002] Collagen is the most abundant protein in animals and a major component of tissues such as skin, bones, tendons, and teeth. Collagenase is an enzyme that specifically degrades collagen, acting on its chain-like structure to cleave interchain bonds and hydrolyze it into low-molecular-weight collagen peptides, including the much-discussed collagen tripeptide. The typical structure of collagen tripeptide is "Gly-XY," where X is mostly proline and Y is mostly hydroxyproline. Collagen tripeptides have wide applications and have become a research hotspot in the health and wellness field, used for anti-wrinkle moisturizing, repairing damaged skin, improving bone and joint health, tissue engineering materials, wound healing promoters, and more.

[0003] Traditional collagenase production primarily relies on extraction from animal tissues (such as the pancreas and stomach). This method is complex, costly, and carries potential risks of disease transmission, limiting its large-scale industrial application. To overcome these limitations, researchers have recently begun exploring microbial fermentation for collagenase production. Microbial fermentation offers readily available raw materials, low cost, and ease of control, making it an important direction for collagenase production. While this method holds great potential, it also faces challenges in practical applications, such as low enzyme activity and high production costs. Therefore, methods such as screening for highly active collagenase mutants and mutants with high substrate binding specificity are needed to promote efficient enzyme production and application.

[0004] Therefore, developing collagenases with excellent enzymatic hydrolysis properties is of great research value. Summary of the Invention

[0005] In order to solve the above-mentioned problems in the prior art, the present invention aims to provide a collagenase mutant, gene fragment, recombinant plasmid, recombinant expression system and its uses, and a method for preparing collagenase mutant and collagen tripeptide.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The present invention provides a collagenase mutant, wherein the amino acid sequence of the collagenase mutant has at least 99% sequence identity with the sequence shown in SEQ ID NO:3.

[0008] Furthermore, the amino acid sequence of the collagenase mutant is shown in SEQ ID NO:3.

[0009] The present invention also provides a gene fragment encoding a collagenase mutant, the nucleotide sequence of which is shown in SEQ ID NO:4 or SEQ ID NO:6.

[0010] The present invention also provides a recombinant plasmid comprising the gene fragments described above.

[0011] The present invention also provides a recombinant expression system containing a host bacterium and the above-mentioned recombinant plasmid.

[0012] The present invention also provides a method for preparing the above-mentioned collagenase mutant, the method comprising the following steps:

[0013] (1) The DNA fragment shown in SEQ ID NO:4 or SEQ ID NO:6 is cloned into the vector plasmid and transformed into E. coli to obtain the expression plasmid;

[0014] (2) The expression plasmid was transferred into the host bacteria, the colony spores were collected, cultured, and the fermentation supernatant enzyme solution was collected to obtain the collagenase mutant.

[0015] Further, the vector plasmid in step (1) is pSET152, and the Escherichia coli is DH5α; the host bacterium in step (2) is Streptomyces lividans TK24; the transfer method is conjugative transfer.

[0016] The present invention also provides the use of the above-mentioned collagenase mutant, gene fragment, recombinant plasmid, and recombinant expression system in the preparation of collagen tripeptides.

[0017] The present invention also provides a method for preparing collagen tripeptides, the method comprising the following steps: using collagen as raw material, sequentially hydrolyzing it with the above-mentioned collagenase mutant and chymotrypsin to obtain collagen tripeptides.

[0018] Furthermore, the chymopapain is papain.

[0019] The present invention has achieved the following beneficial effects:

[0020] This invention provides a collagenase mutant, KT231, that achieves efficient secretory expression in *Streptomyces lividans* TK24. The collagenase mutant KT231 can efficiently hydrolyze Alaska pollock skin at 60°C, exhibiting excellent heat resistance and high enzyme activity, and can be used for the efficient preparation of collagen tripeptides from Alaska pollock skin. When combined with commercially available papain, it significantly increases the proportion of collagen tripeptides in the fish skin hydrolysate without a bitter taste. It shows broad application prospects in the preparation of anti-wrinkle moisturizing products, products for repairing damaged skin, improving bone and joint health, tissue engineering materials, and wound healing promoters.

[0021] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0022] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0023] Figure 1 shows the SDS-PAGE images of collagenases KT16 and KT231.

[0024] Figure 2 shows the pH curves of collagenases KT16 and KT231.

[0025] Figure 3 shows the temperature curves of collagenases KT16 and KT231.

[0026] Figure 4 shows the enzyme activity curves of collagenases KT16 and KT231.

[0027] Figure 5 shows the liquid chromatograms of oligopeptides of different molecular weights in the enzymatic hydrolysate of Alaska pollock skin.

[0028] Figure 6 is a liquid chromatogram of collagen tripeptide content in the enzymatic hydrolysate of Alaska pollock skin. Detailed Implementation

[0029] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0030] The culture medium formulations involved in the embodiments of this invention are as follows: % refers to mass percentage, and g / L refers to grams per liter:

[0031] LB liquid medium: 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride;

[0032] LB solid medium: 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar;

[0033] Skim milk powder culture medium: 1% tryptone, 0.5% yeast extract, 0.5% sodium chloride, 1% skim milk powder, 1.5% agar;

[0034] MS solid medium: soybean flour 20.0 g / L, mannitol 20.0 g / L, calcium carbonate 3.0 g / L, agar 20.0 g / L;

[0035] TSB medium for Streptomyces seed culture: 1.5% tryptone, 0.5% soybean peptone, 0.5% sodium chloride;

[0036] Streptomyces fermentation medium: glucose 10.0 g / L, tryptone 5.0 g / L, yeast extract 5.0 g / L, casein amino acids 2.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, magnesium sulfate heptahydrate 0.5 g / L.

[0037] Example 1: Gene Cloning and Expression Plasmid Construction

[0038] Construction of the recombinant expression plasmid pSET152-KT16 for wild-type collagenase KT16: The expression cassette DNA fragment of the gene kt16 was chemically synthesized, as shown in SEQ ID NO.5, containing the Streptomyces strong promoter KasO*p, the full-length coding sequence of the gene kt16 (collagenase-kt16), and the strong terminator Ter-SCO0356. The amino acid sequence of collagenase-kt16 is shown in SEQ ID NO.1; the coding nucleotide sequence of the gene kt16 is shown in SEQ ID NO.2.

[0039] Plasmid pSET152 was digested with restriction endonuclease BamHI, and the linearized pSET152 DNA was recovered by gel electrophoresis. The two DNA fragments (i.e., the expression cassette DNA fragment of gene kt16 and the linearized pSET152 DNA) were seamlessly cloned and transformed into E. coli DH5α. Plasmids were extracted from positive clones and sequenced to obtain the correct expression plasmid pSET152-KT16.

[0040] Construction of the recombinant expression plasmid pSET152-KT231 for the collagenase mutant KT231: The expression cassette DNA fragment of the gene kt231 was chemically synthesized, as shown in SEQ ID NO.6, containing the Streptomyces strong promoter KasO*p, the full-length coding sequence of the gene kt231 (collagenase-kt231), and the strong terminator Ter-SCO0356. The amino acid sequence of collagenase-kt231 is shown in SEQ ID NO.3; the coding nucleotide sequence of the gene kt231 is shown in SEQ ID NO.4.

[0041] Plasmid pSET152 was digested with restriction endonuclease BamHI, and the linearized pSET152 DNA was recovered by gel electrophoresis. The two DNA fragments (i.e., the expression cassette DNA fragment of gene kt231 and the linearized pSET152 DNA) were seamlessly cloned and transformed into E. coli DH5α. Plasmids were extracted from positive clones and sequenced to obtain the correct expression plasmid pSET152-KT231.

[0042] The target DNA sequences shown in SEQ ID NO.5 and SEQ ID NO.6 provided in the examples can be obtained by PCR amplification and chemical synthesis, and the examples are not limited thereto.

[0043] Example 2: Heterologous expression of collagenase and preparation of fermentation supernatant enzyme solution

[0044] Streptomyces conjugation transfer donor host strain E. coli ET12567 / pUZ8002 was activated on LB agar plates containing 25 μg / mL kanamycin and 25 μg / mL chloramphenicol. The expression plasmids pSET152-KT16 and pSET152-KT231 provided in the above examples were transformed into E. coli ET12567 / pUZ8002, respectively. Positive clones were screened on plates containing 100 μg / mL apramycin, 25 μg / mL kanamycin, and 25 μg / mL chloramphenicol. Then, positive clones containing expression plasmids pSET152-KT16 or pSET152-KT231 were transformed into Streptomyces *S. lividans* TK24 via conjugation transfer. The Mg2+ used in the conjugation transfer process... 2+The concentration was 10 mmol / L, the pre-germination time of Streptomyces was 3 h, the final concentration of apramycin was 1.25 mg / mL, and the final concentration of naphthiopicryl acid was 0.5 mg / mL. After conjugation transfer, MS plates were incubated statically at 28 °C. After 5-8 days, single-clonal conjugators were picked and passaged once on medium containing 25 mg / L naphthiopicryl acid and 100 μg / mL apramycin to obtain Streptomyces expression strains of wild-type collagenase KT16 and mutant KT231.

[0045] Freshly activated *Streptomyces* strains expressing wild-type collagenase KT16 and its mutant KT231 were collected and inoculated into 30 mL of TSB seed culture medium, respectively, and cultured at 30 °C and 220 rpm for 24 h. 3 mL of the seed culture was transferred to 50 mL of fermentation medium and cultured at 30 °C and 220 rpm for 96 h. The supernatant was collected by centrifugation at 8000 g for 10 min to obtain proteins KT16 and KT231. The molecular weights of proteins KT16 and KT231 were analyzed by SDS-PAGE, and the results are shown in Figure 1.

[0046] Referring to GB / T 23527.1-2023 "Quality Requirements for Enzyme Preparations Part 1: Protease Preparations", the degradative enzyme activities of collagenase KT16 and collagenase mutant KT231 on the substrate casein were determined by spectrophotometry at 45℃ and pH 9.0. Enzyme activity is defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute under specific conditions, which is one unit of enzyme activity. The expression activity of wild-type enzyme KT16 was 5231.56 U / mL, while the expression activity of mutant enzyme KT231 was 6602.12 U / mL, an increase of 1.26 times. The above results indicate that the 4-site mutant KT231 obtained in this application has a significantly higher secretory expression level than wild-type enzyme KT16, which is beneficial for further improving enzymatic hydrolysis efficiency and reducing application costs.

[0047] The following experimental examples demonstrate the beneficial effects of the present invention.

[0048] Experimental Example 1: pH stability of the mutant enzyme of this invention

[0049] 50 mmol / L borate-borax-sodium hydroxide buffer solutions with pH values ​​of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, and 12.0 were prepared. Collagenase KT16 and KT231 enzyme solutions were diluted to 10-15 U / mL using these buffer solutions at different pH values. The enzyme activities of collagenase KT16 and KT231 on the substrate casein were measured at 50℃ and the corresponding pH conditions. The highest enzyme activity was defined as 100%, and the corresponding pH was the optimal pH for enzyme hydrolysis. The relative enzyme activities under other pH conditions were calculated. The results are shown in Figure 2. The optimal pH for wild-type KT16 and mutant KT231 on the substrate casein was 8.5. Both are alkaline proteases, and the 4-site mutation did not significantly affect the pH properties of the enzymes.

[0050] Experimental Example 2: Thermostability of the Mutant Enzyme of the Present Invention

[0051] Based on the optimal operating pH determined in Experiment Example 1, the casein hydrolysis activities of collagenases KT16 and KT231 were measured at pH 8.5 at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃.

[0052] As shown in Figure 3, the optimal enzymatic hydrolysis temperature of wild-type enzyme KT16 is 40℃, while the optimal enzymatic hydrolysis temperature of mutant enzyme KT231 is increased to 60℃. This indicates that the 4-site mutant KT231 has better heat resistance and is more suitable for high-temperature enzymatic hydrolysis conditions in industrial practice to obtain higher enzymatic hydrolysis efficiency.

[0053] Experimental Example 3: Collagenase hydrolysis performance of the mutant enzyme of the present invention

[0054] Under high temperature conditions, fish skin collagen is converted into soluble gelatin, and the activity of collagenase can be determined using gelatin as a substrate. Following Zhang's method (Zhang Y, Fu Y, Zhou S, et al. Astraightforward ninhydrin-based method for collagenase activity and inhibitor screening of collagenase using spectrophotometry[J].AnalyticalBiochemistry,2013,437(1):46-48.), the activities of enzymes KT16 and KT231 on gelatin substrate were determined using the ninhydrin colorimetric method. One enzyme activity unit (U) is defined as the amount of enzyme required to degrade one milligram of gelatin per minute to produce 1 μmol of glycine under specific conditions.

[0055] The gelatin-degrading enzyme activities of KT16 and KT231 were determined within a temperature range of 30℃-80℃. The enzyme activity determination method at 30℃ is as follows: A 2 mg / mL gelatin solution was prepared using a Tris·HCl buffer solution (50 mmol / L Tris·HCl, 5 mmol / L CaCl2, pH 8.5) at pH 8.5. 100 μL of the fermentation supernatant of KT16 and KT231 prepared according to the steps in Example 2 was taken, and 2.5 mL of the gelatin solution was added. The mixture was stirred and reacted at 30℃ for 1 h. The reaction was then terminated by adding 2.5 mL of stop solution (25 mmol / L EDTA, 12% (w / v) PEG6000). 100 μL of the reaction solution after termination was added to 900 μL of ultrapure water and mixed with 2.5 mL of ninhydrin colorimetric solution. The mixture was boiled and heated for 10 min. After cooling, 3 mL of ultrapure water was added, and the mixture was stirred thoroughly. The absorbance was measured at 570 nm. The blank control group consisted of 100 μL of boiled and inactivated diluted enzyme solution reacted with 2.5 mL of gelatin solution, with other steps synchronized with the experimental group. 100 μL of glycine solutions of different concentrations were reacted with ninhydrin-sodium citrate solution, and a standard curve was plotted using the absorbance at 570 nm.

[0056] The method for determining the gelatin-degrading enzyme activity under conditions of 35℃-80℃ is the same as described above.

[0057] The results are shown in Figure 4. The enzymatic hydrolytic activity of the enzyme on gelatin substrate showed the same trend as that on casein substrate. At pH 8.5 and 40℃, enzyme KT16 exhibited the highest enzymatic hydrolytic activity on gelatin, while the optimal hydrolytic temperature for KT231 was increased to 60℃. Furthermore, at the corresponding optimal hydrolytic temperatures, the collagenase activity of the mutant enzyme KT231 was 9254.62 U / mL, while that of the wild-type enzyme KT16 was 7238.31 U / mL. The mutant enzyme was significantly higher than the wild-type enzyme, being 1.28 times higher. These results further confirm that the mutant enzyme KT231 is more suitable for the enzymatic hydrolysis of collagen under high-temperature conditions, better meeting the requirements for industrial applications.

[0058] Experimental Example 4: Preparation of collagen tripeptides by enzymatic hydrolysis of Alaska pollock skin using the mutant enzyme of this invention.

[0059] Freshly pretreated Alaska pollock skin was rinsed thoroughly with running water, cut into small pieces, and placed in a food processor. Four to five times the volume of water was added, and the skin was pulverized into a smooth, uniform paste. The mixture was heated to 60°C, and the pH was adjusted to 8.5. Collagenase mutant KT231 was added at an enzyme / substrate ratio of 400 U / g, and the mixture was stirred at 60°C for 2 hours. After the above reaction, the pH of the reaction solution was adjusted to 6.0 with phosphoric acid. Papain was added at an enzyme / substrate ratio of 400 U / g, and the mixture was stirred at 60°C for 2 hours. The reaction solution was then heated to 90°C for 30 minutes to inactivate the protease, yielding an aqueous solution of collagen tripeptides prepared from Alaska pollock skin through enzymatic hydrolysis (i.e., fish skin hydrolysate). A collagen tripeptide aqueous solution prepared by the combined action of commercially available Novozymes peptidase Alcalase and papain was used as a control.

[0060] The extraction efficiency of Alaska pollock skin collagen was characterized by the concentration ratio of hydroxyproline in the enzymatic hydrolysate. The extraction efficiency was calculated using the following formula:

[0061] Extraction efficiency (%) = m1 × dilution factor × 11.1 / m0 × 100

[0062] In the formula, m1 is the hydroxyproline content in g; 11.1 is the coefficient for replacing hydroxyproline with collagen; and m0 is the total protein content of Alaska pollock skin.

[0063] The total protein content in Alaska pollock skin was determined by the Kjeldahl method according to GB 5009.5-2016, "National Food Safety Standard - Determination of Protein in Food". The hydroxyproline content in the enzymatic hydrolysate was determined according to the method of Guo (Guo Hengbin, Zeng Qingzhu. Spectrophotometric Determination of Hydroxyproline Content in Fish Skin. Food Research and Development, 2007(28):145-147.). A standard curve was plotted with the mass concentration of L-hydroxyproline standard as the x-axis and the absorbance at 560 nm as the y-axis. The regression equation was y = 0.5332x - 0.0007 (R0). 2 =0.99993). Using the above regression equation, calculate the hydroxyproline content in the fish skin hydrolysate prepared by enzyme KT231 or enzyme Alcalase.

[0064] At 60℃, the extraction rate of collagen from pollock skin was 81.14% after enzymatic hydrolysis with Alcalase and papain in the control group; after enzymatic hydrolysis with KT231 and papain, the extraction rate was 96.26%, which was 118.63% of the control group, indicating a significant improvement in the extraction efficiency of pollock skin collagen.

[0065] The molecular weight and proportion of peptides in the enzymatic hydrolysate of Alaska pollock skin were determined by high-performance liquid chromatography (HPLC), and the results are shown in Figure 5 and Table 1. In the enzymatic hydrolysate prepared with Alacase and papain, peptides with a molecular weight less than 1000 Da accounted for 88.56%, and peptides with a molecular weight less than 500 Da accounted for 69.32%. In the enzymatic hydrolysate prepared with KT231 and papain, the proportion of peptides with a molecular weight less than 1000 Da increased to 96.76%; among them, the proportion of peptides with a molecular weight less than 500 Da reached 80.26%, which is 115.8% of the control group.

[0066] The above results indicate that the mutant enzyme KT231 of this invention has a higher enzymatic hydrolysis efficiency for Alaska pollock skin, and the proportion of low molecular weight peptides in the skin hydrolysate is significantly increased, which is beneficial to maintaining the excellent biological activity of the prepared peptides.

[0067] Table 1. Percentage of oligopeptides of different molecular weights in fish skin hydrolysate

[0068] Using collagen tripeptide GPH (glycine-proline-hydroxyproline) as a standard, the content of collagen tripeptide (CTP) in the enzymatic hydrolysate was determined by high performance liquid chromatography (HPLC). The results are shown in Figure 6 and Table 2. In the control group, the collagen tripeptide content in the fish skin enzymatic hydrolysate was 24.13%, while in the fish skin enzymatic hydrolysate prepared with the mutant collagenase KT231, the collagen tripeptide content reached 38.62%, an increase of 1.60 times, achieving a significant improvement. Furthermore, the collagen tripeptide prepared by enzymatic hydrolysis with the mutant collagenase KT231 had no bitter taste.

[0069] Table 2. Content of collagen tripeptides in fish skin enzymatic hydrolysate

[0070] In summary, this invention provides a collagenase mutant, KT231, that achieves efficient secretory expression in *Streptomyces lividans* TK24. The collagenase mutant KT231 can efficiently hydrolyze Alaska pollock skin at 60°C, exhibiting excellent heat resistance and high enzyme activity, and can be used for the efficient preparation of collagen tripeptides from Alaska pollock skin. When combined with commercially available papain, it significantly increases the proportion of collagen tripeptides in the fish skin hydrolysate without a bitter taste. It shows broad application prospects in the preparation of anti-wrinkle and moisturizing products, repair damaged skin, improve bone and joint health, tissue engineering materials, and wound healing promoters.

Claims

1. A collagenase mutant, characterized in that: The amino acid sequence of the collagenase mutant has at least 99% sequence identity with the sequence shown in SEQ ID NO:

3.

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

3.

3. A gene fragment encoding a collagenase mutant, characterized in that: The nucleotide sequence of the gene fragment is shown in SEQ ID NO:4 or SEQ ID NO:

6.

4. A recombinant plasmid, characterized by: The plasmid includes the gene fragment as described in claim 3.

5. A recombinant expression system, characterized in that: The recombinant expression system contains a host bacterium and the recombinant plasmid as described in claim 4.

6. A method of producing the collagenase mutant of claim 1 or 2, comprising, The method includes the following steps: (1) The DNA fragment shown in SEQ ID NO:4 or SEQ ID NO:6 is cloned into the vector plasmid and transformed into E. coli to obtain the expression plasmid; (2) The expression plasmid was transferred into the host bacteria, the colony spores were collected, cultured, and the fermentation supernatant enzyme solution was collected to obtain the collagenase mutant.

7. The method according to claim 6, characterized in that: The vector plasmid mentioned in step (1) is pSET152, and the Escherichia coli is DH5α; the host bacterium mentioned in step (2) is Streptomyces lividans TK24; The transfer method is a combination transfer.

8. Use of the collagenase mutant of claim 1 or 2, the gene fragment of claim 3, the recombinant plasmid of claim 4, and the recombinant expression system of claim 5 in the preparation of collagen tripeptides.

9. A method of preparing a collagen tripeptide, characterized by, The method includes the following steps: using collagen as raw material, it is sequentially hydrolyzed by the collagenase mutant and chymotrypsin as described in claim 1 or 2 to obtain collagen tripeptide.

10. The method of claim 9, wherein: The chymopapain is papain.

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