Collagenase mutant as well as enzyme composition and application thereof

Abstract

The invention belongs to the technical field of collagen peptide preparation, and particularly relates to a collagenase mutant and an enzyme composition and application thereof. The amino acid sequence of the collagenase mutant is as shown in SEQ ID NO. 2. The collagenase mutant (Vow1113) provided by the invention has the advantages of high enzyme activity, wide catalytic temperature range, capability of adapting to a high-temperature production environment of collagenase enzymolysis and the like, and can effectively degrade a collagen raw material from fish skin by matching with alkaline protease and flavourzyme, so that the yield of low-molecular-weight peptide is increased, and the production cost is reduced. The active polypeptide with the molecular weight smaller than 500 Da, the proportion exceeding 80%, the collagen tripeptide proportion exceeding 35% and the GPH content obviously improved is generated, the moisture absorption and moisturizing performance of the obtained collagen peptide is greatly improved, the water holding capacity of the product is improved, and the active polypeptide has good application prospects in the fields of skin care products and oral beauty products.

Description

Technical Field

[0001] This invention relates to the field of collagen peptide preparation technology, and in particular to a collagenase mutant and its enzyme composition and applications. Background Technology

[0002] Collagen tripeptide (CTP) is a small-molecule bioactive peptide obtained from the hydrolysis of collagen. Its typical structure is Gly-XY, where X is mostly proline and Y is mostly hydroxyproline. Due to its small molecular weight, it is easily absorbed and utilized by the human body, exhibits good biocompatibility, low immunogenicity, and various physiological functions, and is trending towards replacing collagen in various applications. Collagen tripeptide possesses excellent moisturizing properties, stemming from its rich content of hydrophilic polar groups such as amino and carboxyl groups. These groups can form hydrogen bonds with water molecules, adsorbing moisture from the environment and maintaining the hydration of the stratum corneum. Simultaneously, it can interact with other biomolecules in the skin to construct a water-retaining network structure, reducing skin moisture loss. Based on this, CTP is commonly used in oral supplements (common product forms include collagen peptide powder and oral liquid) or topical skincare products to help improve skin firmness, elasticity, hydration, and radiance, demonstrating high application value in the cosmetics and food industries. In addition, CTP can promote the synthesis of type II collagen, which helps relieve joint pain and stiffness in patients with osteoarthritis, and shows excellent performance in joint care, wound repair and other medical fields.

[0003] CTP preparation mainly includes chemical methods, microbial fermentation methods, and enzymatic hydrolysis methods. Chemical methods utilize acids and alkalis to hydrolyze collagen; while rapid and low-cost, they easily damage the amino acid structure, affecting product purity and activity. Microbial fermentation uses microorganisms such as Bacillus and Lactobacillus to produce enzymes, which then enzymatically hydrolyze collagen. This method is environmentally friendly and operates under mild conditions; however, microbial metabolic byproducts may affect product quality, requiring precise control of fermentation conditions. Enzymatic hydrolysis uses specific proteases such as pepsin and trypsin to directionally hydrolyze collagen into collagen tripeptides under suitable conditions. This method operates under mild conditions, preserves amino acid structure and activity, and produces products with high safety and purity. By controlling the enzyme dosage, reaction time, and temperature, the degree of hydrolysis can be adjusted to obtain high-quality collagen tripeptides.

[0004] Currently, enzymatic hydrolysis is widely used in industrial production to prepare collagen tripeptides. However, this method suffers from low hydrolysis efficiency and insufficient content of characteristic peptides. For example, the content of low molecular weight collagen peptides (molecular weight less than 500 Da) is low, especially the content of the core tripeptide with the glycine-proline-hydroxyproline (GPH) sequence, which is low and the yield is unstable, affecting the bioactivity of the target product. Therefore, developing novel collagenases with excellent hydrolytic performance is of significant theoretical and practical value for promoting the industrial production and high-value application of collagen tripeptides. Summary of the Invention

[0005] To address the problems of low enzymatic hydrolysis efficiency and low content of characteristic peptides in collagen hydrolysates in existing technologies, this invention, based on a newly isolated collagenase, performs multi-site mutations to obtain a novel collagenase mutant. The invention provides the encoding gene, recombinant expression vector, and recombinant strain of this collagenase mutant, and further provides an enzyme composition containing this collagenase mutant and its application in the preparation of collagen peptides. This invention is specifically achieved through the following technical solutions:

[0006] A first aspect of the present invention provides a collagenase mutant having the amino acid sequence shown in SEQ ID NO.2.

[0007] A second aspect of the present invention provides a nucleic acid molecule that encodes a collagenase mutant as described above.

[0008] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.4.

[0009] A third aspect of the present invention provides an expression vector comprising the nucleic acid molecule described above.

[0010] Furthermore, the expression vector is the Pichia pastoris expression vector pPIC9K.

[0011] A fourth aspect of the present invention provides a recombinant strain comprising the expression vector described above.

[0012] Furthermore, the recombinant strain is Pichia pastoris GS115.

[0013] A fifth aspect of the present invention provides an enzyme composition comprising an alkaline protease, a flavor protease, and a collagenase mutant as described above.

[0014] The sixth aspect of the present invention provides the use of the collagenase mutant or enzyme composition as described above in the preparation of collagen peptides.

[0015] The seventh aspect of this invention provides a method for preparing collagen peptides, comprising the following steps:

[0016] S1. Pre-treat the cleaned fish skin to obtain a crude collagen solution;

[0017] S2. The crude collagen solution is sequentially subjected to alkaline protease, flavor protease and collagenase mutant enzymatic hydrolysis, and then the enzymatic hydrolysis is terminated to obtain enzymatic hydrolysate, wherein the amount of alkaline protease, flavor protease and collagenase mutant is 0.1‰-0.5‰ of the dry weight of fish skin.

[0018] S3. The enzymatic hydrolysate is subjected to deimpurity and deodorization treatment to obtain a purified collagen peptide solution.

[0019] Furthermore, in step S1, the pretreatment includes degreasing and swelling treatment.

[0020] Furthermore, step S2 specifically includes the following steps:

[0021] S21. Add alkaline protease solution at 0.5‰ of the dry weight of fish skin, and stir and react for 3 hours at pH 8.0-9.0 and 50℃.

[0022] S22. Add flavor protease at 0.2‰ of the dry weight of fish skin, and stir and react for 2 hours at pH 7.0-7.5 and 50℃.

[0023] S23. Add collagenase mutant solution at 0.4‰ of the dry weight of fish skin, and stir and react for 3 hours at pH 6.0 and 60℃.

[0024] S24. Raise the temperature of the reaction mixture to 90°C and heat for 30 min. Then centrifuge to collect the intermediate liquid to obtain the enzymatic hydrolysate.

[0025] Further, step S3 specifically includes the following steps: adding activated carbon at 0.5% of the dry weight of fish skin, adsorbing at 40°C for 1 hour, centrifuging to remove activated carbon, collecting the supernatant, and filtering and separating it using a nanofiltration ceramic membrane with a retention range of less than or equal to 5000 Da to obtain a purified collagen peptide solution.

[0026] The advantages and positive effects of this invention are as follows:

[0027] The collagenase mutant (Vow1113) provided by this invention has advantages such as high enzyme activity, wide catalytic temperature range, and adaptability to the high-temperature production environment of collagenase hydrolysis. By combining with alkaline protease and flavor protease, it can effectively degrade collagen raw materials from fish skin, increase the yield of low molecular weight peptides, and generate active peptides with a molecular weight <500 Da of more than 80%, a collagen tripeptide content of more than 35%, and a significantly increased GPH content. This greatly improves the hygroscopic and moisturizing properties of the obtained collagen peptides, enhances the water-holding capacity of the products, and has good application prospects in the fields of skin care products and oral beauty products. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 SDS-PAGE electrophoresis images of the fermentation enzyme broth of recombinant wild-type collagenase Vow101 and its mutant Vow1113 in this embodiment of the invention.

[0030] Figure 2 This is a graph showing the results of the optimal temperature and optimal pH determination for wild-type collagenase Vow101 and its mutant Vow1113 in the embodiments of the present invention.

[0031] Figure 3 The high-performance liquid chromatograms of GPH and standards in the enzymatic hydrolysate treated with wild-type collagenase Vow101 and its mutant Vow1113 in this embodiment of the invention are shown.

[0032] Figure 4 This is a graph showing the trend of the moisturizing rate of collagen peptides in this invention over time under environmental conditions with relative humidity of 81% and 43%.

[0033] Figure 5 This is a graph showing the trend of moisture absorption rate of collagen peptides in an environment with relative humidity of 81% and 43% over time, according to an embodiment of the present invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0035] Based on the information contained in this application, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0036] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0037] Additionally, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art.

[0038] The terms “including,” “contains,” “includes,” “has,” and similar words are non-restrictive and can include other steps and other components that do not affect the result.

[0039] The term “and / or” should be regarded as referring to a specific disclosure of each of the two specified features or components, with or without the other. For example, “A and / or B” would be regarded as including (i) A, (ii) B, and (iii) A and B.

[0040] The term "gene" is the complete nucleotide sequence required to produce a polypeptide chain or functional RNA. A gene can contain several operablely linked nucleic acid segments, such as a 5' non-coding region, a coding sequence, and a 3' non-coding region containing polyadenylation sites. Non-coding regions generally have regulatory functions on gene expression, such as promoters and terminators.

[0041] The term "vector" refers to a self-replicating DNA molecule that transfers a target gene into a host cell, and it is often in the form of a circular double-stranded DNA molecule. A vector containing a foreign gene is called a recombinant vector.

[0042] The term "expression vector" allows a target gene inserted into a vector to be expressed in a host cell, containing regulatory elements for expression in the designated host cell, such as promoters and / or terminators. An expression vector is introduced into an appropriate host cell to enable it to express the inserted target gene.

[0043] The terms "import" or "transfer" refer to the transfer of a target gene into a host cell, resulting in stable genetic inheritance. The imported nucleic acid molecule can be in plasmid form retained in the host cell or can be integrated into the host cell genome. Nucleic acid molecules and / or vectors can be transferred into host cells via methods such as "transfection," "transformation," or "transduction." Host cells containing the imported nucleic acid molecule are referred to as "transgenic," "recombinant," "transformed," or "engineered" organisms. Vector introduction into host cells can be performed using conventional techniques well known to those skilled in the art.

[0044] Unless otherwise stated, terms such as "nucleotide," "nucleic acid," "nucleic acid molecule," and "nucleic acid fragment" are interchangeable in the context of this invention. The terms "gene," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid molecule" as used in this invention refer to a polymeric form of nucleotides of any length, which may be ribonucleotides or deoxyribonucleotides; this term refers only to the primary structure of the molecule.

[0045] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below.

[0046] Currently, the enzymatic hydrolysis preparation process of collagen tripeptide (CTP) has problems such as a limited variety of collagenases, low hydrolysis efficiency of some enzymes, and insufficient content of characteristic peptides, especially low content of low molecular weight peptides, which affect the industrial application of collagen hydrolysate and collagen tripeptide.

[0047] To expand the types of collagenases and improve their activity, this invention uses wild-type Vibrio owensii isolated from sand and mud in the nearshore aquaculture area of ​​Hainan as the starting strain. Based on whole-genome sequencing data, a novel collagenase, Vow101 (amino acid sequence shown in SEQ ID NO.1), was isolated. Based on its wild-type sequence, the target protein was predicted with high precision in three dimensions using AlphaFold2 software. Using structural visualization software such as PyMOL and UCSF Chimera, combined with molecular docking technology, the spatial interactions between the mutation site and the protein's active site, substrate-binding pocket, and key functional domains were analyzed. Structural homology modeling and comparison were performed using tools such as I-TASSER and Swiss-Model to predict candidate mutation sites with significant potential for improving protein stability, enzyme activity, or stress resistance. Site-directed mutagenesis was performed on the candidate mutation sites. The mutant gene was secreted and expressed in the Pichia pastoris system (vector pPIC9K, host strain GS115). The hydrolytic ability and activity of the recombinant strain's fermentation enzyme on gelatin substrate were detected to verify the target trait and screen for the mutant Vow1113. A positive modified mutant with enhanced catalytic activity, Vow1113 (amino acid sequence shown in SEQ ID NO.2), was obtained through screening.

[0048] Compared to wild-type collagenase Vow101, the collagenase mutant Vow1113 contains the following 12 site changes: amino acid 19 changes from S to N (S19N), amino acid 29 changes from I to V (I29V), amino acid 46 changes from R to S (R46S), amino acid 155 changes from R to K (R155K), amino acid 212 changes from S to N (S212N), amino acid 238 changes from T to A (T238A), amino acid 246 changes from V to A (V246A), amino acid 272 changes from S to R (S272R), amino acid 429 changes from V to A (V429A), amino acid 541 changes from R to Q (R541Q), amino acid 695 changes from V to A (V695A), and amino acid 728 changes from L to V (L728V). In terms of enzymatic properties, the mutant Vow1113 exhibits the following characteristics:

[0049] (1) The enzyme activity was significantly increased. The mutant was expressed in a heterologous extracellular environment with gelatin as a substrate. The enzyme activity of the fermentation enzyme liquid of mutant Vow1113 was 3325.51 U / mL, which was 49% higher than that of wild-type collagenase Vow101.

[0050] (2) Improved temperature tolerance: The mutant Vow1113 has higher enzyme activity in the range of 40-70℃, with an optimal temperature of 60℃. Compared with the wild-type collagenase Vow101, which has an optimal temperature of 40℃, its heat resistance is significantly improved, making it more suitable for the high-temperature production conditions of fish skin collagenase hydrolysis.

[0051] (3) It exhibits excellent collagen hydrolysis activity. Fish skin collagen was sequentially treated with alkaline protease, flavor protease, and the mutant Vow1113. The collagen peptides obtained by enzymatic hydrolysis had a molecular weight of less than 500 Da, accounting for 80.17% of the peptides, and the collagen tripeptide content reached 36.53%, of which Gly-Pro-Hyp (GPH) was 3.14%. This significantly increased the content of low molecular weight peptides (<500 Da) and collagen tripeptides, and also increased the content of the characteristic peptide GPH. This is of great significance for improving the biological activity of collagen hydrolysate. Tests showed that in environments with relative humidity of 81% and 43%, the moisturizing and hygroscopic properties of the collagen peptides prepared in this invention were superior to those of glycerol and hyaluronic acid.

[0052] Based on this, an embodiment of the present invention provides a collagenase mutant, the amino acid sequence of which is shown in SEQ ID NO.2.

[0053] The collagenase mutant (Vow1113) provided by this invention has advantages such as high enzyme activity, wide catalytic temperature range, and adaptability to the high-temperature production environment of collagenase hydrolysis. By combining with alkaline protease and flavor protease, it can effectively degrade collagen raw materials from fish skin, increase the yield of low molecular weight peptides, and generate active peptides with a molecular weight <500 Da of more than 80%, a collagen tripeptide content of more than 35%, and a significantly increased GPH content. This greatly improves the hygroscopic and moisturizing properties of the obtained collagen peptides, enhances the water-holding capacity of the products, and has good application prospects in the fields of skin care products and oral beauty products.

[0054] Another embodiment of the present invention provides a nucleic acid molecule encoding a collagenase mutant as described above.

[0055] The advantages of the nucleic acid molecule over the prior art are the same as those of the collagenase mutant over the prior art as described above, and will not be repeated here.

[0056] Nucleic acid molecules include DNA molecules (such as genomic DNA or cDNA) and / or RNA molecules (such as mRNA), and can be single-stranded or double-stranded. The sequence of a nucleic acid molecule can be derived from the amino acid (AA) sequence of a collagenase mutant using conventional methods such as codon coding rules. The full-length sequence of a nucleic acid molecule or a fragment thereof can usually be obtained using PCR amplification, recombination, or artificial synthesis.

[0057] For example, the gene sequence encoding the collagenase mutant is shown in SEQ ID NO.4.

[0058] Those skilled in the art will understand that, due to the degeneracy of the genetic code, gene sequences different from the examples above can also encode the collagenase mutant of the present invention. Therefore, the sequences in the examples above should not be construed as limiting the scope of protection of the present invention.

[0059] Another embodiment of the present invention provides an expression vector comprising the nucleic acid molecules described above.

[0060] The advantages of the expression vector over the prior art are the same as those of the collagenase mutant over the prior art as described above, and will not be repeated here.

[0061] Optionally, the expression vector further includes gene sequences encoding a signal peptide, a promoter, and / or a terminator. The collagenase mutant is located downstream of the signal peptide, the promoter is located upstream of the signal peptide, and the terminator is located downstream of the collagenase mutant. That is, when all the aforementioned expression elements are included, the gene connection sequence from upstream to downstream is: promoter—signal peptide—collagenase mutant—terminator. The signal peptide is used to guide the secretory expression of collagenase in the host bacteria, facilitating isolation and purification. The promoter is used to initiate the transcription process of the gene, and the terminator is used to terminate the transcription of the gene to form a complete collagenase expression cassette. The signal peptide, promoter, and terminator are adaptively selected according to the type of expression vector and host cell; and in actual recombinant expression, each expression element can also be selected according to actual needs. For example, if secretory expression is not required or the selected vector has a signal peptide, it is not necessary to add an additional signal peptide gene upstream of the gene.

[0062] Optionally, the expression vector includes a prokaryotic expression vector, a eukaryotic expression vector, or a viral expression vector (such as lentivirus or adenovirus). Correspondingly, the host cells transformed or transfected by the expression vector can be prokaryotic cells or eukaryotic cells, selected according to the type of expression vector. For example, when using a prokaryotic expression vector, prokaryotic cells are selected as the host cells. Common examples of prokaryotic cells include Escherichia coli, Bacillus, Corynebacterium, *Fermentosum motilityum*, and *Streptomyces*. When using a eukaryotic expression vector, eukaryotic cells are selected as the host cells. Common examples of eukaryotic cells include *Saccharomyces cerevisiae*, *Pichia pastoris*, *Saccharomyces cerevisiae*, and filamentous fungi.

[0063] Typical vectors include plasmids (such as the pUC series, pET series, pWB series, pGEX series, pDXW series, pPIC series, pBR series, and pEZ series), viral vectors, bacteriophages (such as λgt4λB, λ-Charon, λΔz1, and M13), granulocytes, and mini-chromosomes. Plasmids are the most commonly used vectors; therefore, in the context of this invention, unless otherwise specified, plasmids and vectors are used interchangeably.

[0064] Pichia pastoris, as an important protein expression host, has advantages such as mature culture conditions, convenient genetic manipulation, strong metabolic activity, and high protein secretion efficiency. In a typical embodiment of this invention, a Pichia pastoris expression system is used, and the expression vector used is the Pichia pastoris expression vector pPIC9K, which contains the strong promoter P. aox1 (Methanol-induced) secreted signal peptide SP α-factor Histidine deficiency (His⁻) and the genetic mycotoxin resistance gene G418, terminator T aox1 The host (or recipient) cell was Pichia pastoris GS115. A recombinant expression vector was constructed by placing the collagenase gene sequence between the pPIC9K multiple cloning sites. After transformation of Pichia pastoris with this recombinant expression vector, secretory expression of collagenase was achieved through conventional fermentation culture. The target enzyme content in the fermentation supernatant was higher than 95%, therefore the fermentation enzyme solution can be directly used as a high-quality enzyme source for the catalytic reaction of collagen substrates. This is beneficial for improving the production efficiency of pure enzymes and reducing the industrial production cost of collagen peptides.

[0065] In optional embodiments, the expression vector may also be pPIC9, pPIC3K, pPIC3.5K, pPICZA, pPICZB, pGAPZ, pGAPZa, pHIL-S1, pHIL-D2, pA0815, etc.; the Pichia pastoris host strain may also be other yeast strains such as X33, KM71H, SMD116, etc.

[0066] In a preferred embodiment of the present invention, the method for inserting a nucleic acid molecule into an expression vector includes: synthesizing the above-mentioned nucleic acid molecule through whole-genome synthesis, designing first and second homologous arm sites at both ends of the nucleic acid molecule; treating the expression vector with an endonuclease to obtain a linearized vector; and ligating the nucleic acid molecule and the linearized vector using seamless cloning technology to obtain a recombinant expression vector.

[0067] Optionally, the restriction enzyme is EcoRI. The vector homologous arms are adaptively selected based on the EcoRI insertion site, and this invention does not impose any special limitations on this.

[0068] In another embodiment of the present invention, a recombinant strain is provided, the recombinant strain containing the nucleic acid molecule as described above or the expression vector as described above.

[0069] Optionally, the recombinant strain originates from Pichia pastoris, specifically Pichia pastoris GS115.

[0070] Nucleic acid molecules or expression vectors can be transformed or transfected into host strains by various methods known in the art, including: CaCl2 transformation, lithium acetate transformation, calcium phosphate-DNA coprecipitation, electroporation, gene gun bombardment, microinjection, conjugation transfer, liposome-mediated transfection, liposome fusion, lipid transfection, and protoplast fusion, etc.

[0071] This invention also provides a method for preparing collagenase mutants, comprising the following steps:

[0072] The recombinant strain described above is cultured, the fermentation broth is centrifuged, the supernatant is filtered, and the filtrate is collected to obtain the enzyme solution containing the collagenase mutant.

[0073] Optionally, culturing the recombinant strain as described above includes: inoculating the recombinant strain into BMMY liquid medium, culturing at 30°C and 220 r / min, adding methanol at 2% of the total volume of the medium at 24 h and 48 h to induce the secretory expression of the target gene, and collecting the fermentation broth at 72 h.

[0074] The present invention further provides an enzyme composition comprising the collagenase mutant as described above. Specifically, the enzyme composition comprises an alkaline protease, a flavor protease, and a collagenase mutant.

[0075] The above-mentioned enzyme composition is combined with different enzyme cleavage sites. Each protease is used to enzymatically hydrolyze the collagen raw material from fish skin step by step. After adsorption by activated carbon and filtration by ceramic membrane to remove impurities, collagen peptides rich in collagen tripeptides can be obtained. These collagen peptides have excellent moisturizing and hygroscopic properties and can be used in skin care products and oral beauty products to effectively improve skin moisturizing ability and skin moisture content, and improve skin condition.

[0076] Optionally, the alkaline protease is enzyme KT124, the amino acid sequence of which can be found in patent "CN119799680A A protease mutant with a wide pH range and its use in preparing collagen peptides (publication date: 2025-04-11)".

[0077] The embodiments of the present invention also provide the application of the collagenase mutant or its enzyme composition as described above in the preparation of collagen peptides, and provide related preparation methods.

[0078] The preparation method of collagen peptides in this embodiment of the invention includes the following steps:

[0079] S1. Pre-treat the cleaned fish skin to obtain a crude collagen solution;

[0080] S2. The crude collagen solution is sequentially subjected to alkaline protease, flavor protease and collagenase mutant enzymatic hydrolysis, and then the enzymatic hydrolysis is terminated to obtain enzymatic hydrolysate, wherein the amount of alkaline protease, flavor protease and collagenase mutant is 0.1‰-0.5‰ of the dry weight of fish skin.

[0081] S3. The enzymatic hydrolysate is subjected to deimpurity and deodorization treatment to obtain a purified collagen peptide solution.

[0082] Optionally, the pretreatment in step S1 includes defatting and swelling. More specifically, the pretreatment includes the following steps: adding fish skin (after removing fish meat, bones, and connective tissue) to an organic solvent and soaking it at room temperature until the fat is dissolved; then separating the fish skin by filtration; adding the defatted fish skin to clean water and keeping it at 70-80°C for 30 minutes to allow the gelatin in the fish skin to fully swell, resulting in a crude collagen solution.

[0083] Optionally, step S2 specifically includes the following steps:

[0084] S21. Add alkaline protease solution at 0.5‰ of the dry weight of fish skin, and stir and react for 3 hours at pH 8.0-9.0 and 50℃.

[0085] S22. Add flavor protease at 0.2‰ of the dry weight of fish skin, and stir and react for 2 hours at pH 7.0-7.5 and 50℃.

[0086] S23. Add collagenase mutant solution at 0.4‰ of the dry weight of fish skin, and stir and react for 3 hours at pH 6.0 and 60℃.

[0087] S24. Raise the temperature of the reaction mixture to 90°C and heat for 30 min. Then centrifuge to collect the intermediate liquid to obtain the enzymatic hydrolysate.

[0088] Optionally, step S3 specifically includes the following steps: adding activated carbon at 0.5% of the dry weight of fish skin, adsorbing at 40°C for 1 hour, performing decolorization, impurity removal, and deodorization treatment, centrifuging to remove activated carbon, collecting the supernatant, and filtering and separating it using a nanofiltration ceramic membrane with a retention range of less than or equal to 5000 Da to obtain a purified collagen peptide solution.

[0089] The invention is further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer. The main materials in the following embodiments include:

[0090] 1) Biomaterials:

[0091] Gelatin (purchased from Merck, catalog number G7041), aprotinin (purchased from Merck, catalog number Y0001154), bacitracin (purchased from Shanghai Aladdin, catalog number B105485), tetrapeptide GGYR and tripeptide GGG (synthesized by Nanjing Peptide Biotechnology Co., Ltd.), flavor protease Flavorzyme (purchased from Novozymes, Flavorzyme 500MG), expression vector pPIC9K (purchased from Invitrogen, catalog number V17520), TSK gel G2000 SWXL column and C18 reversed-phase column were all commercially available.

[0092] 2) Culture medium:

[0093] (a) MD medium: 13.4 g / L yeast nitrogen base (YNB), 20 g / L glucose, 0.4 mg / L biotin, 20 g / L agar powder

[0094] (b) BMGY liquid medium: 10 g / L yeast extract, 20 g / L peptone, 13.4 g / L yeast nitrogen base (YNB), 0.4 mg / L biotin, 10 g / L glycerol and 0.1 M phosphate buffer (pH 6.0).

[0095] (c) BMMY liquid medium: replace 10 g / L glycerol in (b) with 20 g / L methanol.

[0096] 1. Cloning of wild-type and mutant collagenase genes

[0097] This invention isolated a strain of *Vibrio owensii* from the sand and mud of aquaculture areas in the coastal waters of Hainan. A novel collagenase, named Vow101, was identified in its genome, suitable for preparing collagen tripeptides by hydrolyzing fish skin. Based on this wild-type sequence, key active sites were analyzed using biological analysis software, including three-dimensional result prediction, molecular docking, and homology modeling comparison, to screen key candidate mutation sites and candidate mutant enzymes and their genes. *Pichia pastoris* (P. pastoris) GS115 was used as the host strain to express the candidate mutant gene. After inducing the secretion and expression of the target gene, the cell culture supernatant was collected as the fermentation enzyme solution. By comparing the enzyme activity and enzymatic properties of the mutant fermentation enzyme solution, a collagenase mutant with improved enzyme activity and temperature tolerance was screened and named Vow1113.

[0098] The amino acid sequence of wild-type collagenase Vow101 is shown below:

[0099] lsepsqqvteiyqhhahq s gndralpdy i ptkllpqqpkptlrtl r trsaeqaanvcdveaftsnssndvlnaiktqgascvnalfsaesriqeaafesghmyniakhttdlvkayagggsdelealflylragyyaefynskvsflswvtpav r eavdafvnnanfyensdphgkvlseviitmdsaglqhaylpqitqwltrwdsqyaq s wymrnavngvftilfggqwneqfvq t ignqtel v kalgdfalrssaigasdefmaanag s elgrltkysgsasstvksklteifaqyemygrgdaiwlgaadtvsyyadcsdygicnfesqlkglvlsqsytcsptirilsqnmtqdqhvaacskmgyeegyfhtsletgrqpvadyntqlqvnifdssddygkyagpifnistnnggmylegdp vtpgnipnfvayeapyanpdhfvwnleheyvhyldgrfdlyggfghpterivwwsegiaeyvskendnqaaidtikdgstftlseifetsydgfdvdriyrwgylavrfmfe r hkddvnqmlietrqgnwanykatinqwailyqsefeqwqqalvsgdapnavitansegkvgesitfssenstdangkivsvlwdfgdgttstqtqpshqygsegqytisltvtdndgltastthsltitaqggsdalpqdcavqskisggrlt v gqaaclatqqtiwlsipavnehtsmaittang l gdlkieysndgwpngsnhhawsdnagnaecitlsnqrnywgyvkvsgefenaaivvdfdtsgcrq (see SEQ ID NO.1), the underlined site is the site to be mutated.

[0100] The amino acid sequence of the collagenase mutant Vow1113 is shown below:

[0101] lsepsqqvteiyqhhahq n gndralpdy v ptkllpqqpkptlrtl s trsaeqaanvcdveaftsnssndvlnaiktqgascvnalfsaesriqeaafesghmyniakhttdlvkayagggsdelealflylragyyaefynskvsflswvtpav k eavdafvnnanfyensdphgkvlseviitmdsaglqhaylpqitqwltrwdsqyaq n wymrnavngvftilfggqwneqfvq a ignqtel a kalgdfalrssaigasdefmaanag relgrltkysgsasstvksklteifaqyemygrgdaiwlgaadtvsyyadcsdygicnfesqlkglvlsqsytcsptirilsqnmtqdqhvaacskmgyeegyfhtsletgrqpvadyntqlqvnifdssddygkyagpifnistnnggmylegdp a tpgnipnfvayeapyanpdhfvwnleheyvhyldgrfdlyggfghpterivwwsegiaeyvskendnqaaidtikdgstftlseifetsydgfdvdriyrwgylavrfmfe q hkddvnqmlietrqgnwanykatinqwailyqsefeqwqqalvsgdapnavitansegkvgesitfssenstdangkivsvlwdfgdgttstqtqpshqygsegqytisltvtdndgltastthsltitaqggsdalpqdcavqskisggrlt a gqaaclatqqtiwlsipavnehtsmaittang v gdlkieysndgwpngsnhhawsdnagnaecitlsnqrnywgyvkvsgefenaaivvdfdtsgcrq (see SEQ ID NO.2), the underlined part indicates the mutation site.

[0102] The collagenase mutant Vow1113, compared to the wild-type collagenase Vow101, contains alterations at the following 12 sites: S19N, I29V, R46S, R155K, S212N, T238A, V246A, S272R, V429A, R541Q, V695A, and L728V.

[0103] The coding genes for Vow101 and Vow1113 are shown in Table 1.

[0104] Table 1. Gene (DNA) sequences of embodiments of the present invention

[0105]

[0106] 2. Recombinant expression of collagenase

[0107] Preparation of expression plasmid pY310 carrying the Vow101 gene: The Vow101 gene (SEQ ID NO.3) fragment with the vector homologous arm was synthesized by Suzhou Genewiz Co., Ltd., and seamlessly ligated with the vector pPIC9K (purchased from Invitrogen, catalog number V17520) linearized with restriction endonuclease EcoRI. The seamless cloning operation was performed according to the instructions of the multi-fragment seamless cloning kit (purchased from Shanghai Sangon Biotech Co., Ltd., catalog number B632218-0040). The ligation product was transformed into Escherichia coli DH5α. The plasmid was extracted from the culture of positive colonies and obtained the recombinant expression plasmid pY310 after sequence verification.

[0108] Preparation of expression plasmid pY313 carrying the Vow1113 gene: The Vow1113 gene (SEQ ID NO.4) fragment with a vector homologous arm was synthesized by Suzhou Genewiz Company and seamlessly cloned and ligated with the vector pPIC9K linearized with restriction endonuclease EcoRI; the ligation product was transformed into Escherichia coli DH5α; the plasmid was extracted from the culture of positive colonies and the recombinant expression plasmid pY313 was obtained by sequencing.

[0109] Using Pichia pastoris GS115 as the host, plasmids pY310 and pY313, linearized with restriction endonuclease SacI, were transformed into Pichia pastoris GS115 by electroporation. The GS115 resuscitation solution was plated on MD solid plates and cultured at 30°C for 72 h to obtain the corresponding recombinant yeast strains.

[0110] 3. Preparation of collagenase enzyme solution and analysis of its enzymatic properties

[0111] Single colonies were picked from the transformation plates corresponding to recombinant expression plasmids pY310 and pY313 and inoculated into 50 mL of BMGY liquid medium, and cultured at 30℃ and 220 r / min for 24 h. Afterwards, the cells were centrifuged at 4℃ and 8000 g for 10 min, the supernatant was discarded, and the bacterial cells were collected. All bacterial cells from each colony were resuspended in 50 mL of BMGY liquid medium and cultured at 30℃ and 220 r / min for another 72 h. At 24 h and 48 h, methanol (v / v) at 2% of the total culture medium volume was added to induce the secretory expression of the target gene. At 72 h, the cells were centrifuged at 8000 g for 10 min, and the supernatant was collected, which was the enzyme solution containing collagenases (Vow101, Vow1113).

[0112] The fermentation enzyme solution was directly loaded onto the sample for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel image is shown in [link to image]. Figure 1The results showed that both the wild-type enzyme Vow101 and the mutant enzyme Vow1113 were successfully secreted and expressed in Pichia pastoris. The concentration of the target protein Vow1113 was 0.45 mg / mL, and the purity was higher than 95%.

[0113] 3.1 Enzyme activity assay

[0114] The activity of collagenase on gelatin substrate was determined by ninhydrin colorimetric method. One enzyme activity unit (U) was defined as the amount of enzyme required per hour to degrade one mg of gelatin to produce 1 µmol of leucine at 40°C and pH 6.0.

[0115] The enzyme activity assay was performed as follows: A 10 mg / mL gelatin solution was prepared using 0.1 mol / L Britton-Robinson buffer (pH 6.0), and the Vow101 and Vow1113 enzyme solutions were diluted to appropriate concentrations using this buffer. 1 mL of gelatin solution and 1 mL of diluted collagenase solution were mixed and incubated at 40°C with shaking for 1 h. The reaction was then terminated by adding 2 mL of pre-cooled 1.25 mol / L trichloroacetic acid (TCA) solution. The reaction solution was centrifuged at 10000 g for 10 min, and 200 µL of the enzymatic digestion solution was taken and mixed with 1 mL of ninhydrin-sodium citrate solution. The mixture was boiled for 20 min. After the reaction solution cooled to room temperature, 5 mL of 50% n-propanol (v / v) solution was added, mixed, and the absorbance at 600 nm was measured using a spectrophotometer. Different concentrations of 200 µL leucine solutions were reacted with ninhydrin-sodium citrate solution, and a standard curve was plotted using the absorbance at 600 nm. The blank control group consisted of 1 mL gelatin solution reacted with 1 mL of boiled and inactivated enzyme solution, with all other steps synchronized with the experimental group.

[0116] At 40°C and pH 6.0, using gelatin as a substrate, the extracellular enzyme activity of wild-type Vow101 was 2232.17 U / mL, and the extracellular enzyme activity of mutant Vow1113 was 3325.51 U / mL, which were significantly higher than those of wild-type, representing a 49% increase in enzyme activity compared to wild-type.

[0117] 3.2 Optimal Temperature

[0118] The activities of collagenases Vow101 and Vow1113 were determined at pH 6.0 and different temperatures (30-70℃). The method for determining the activity of gelatin-degrading enzymes was the same as described above. Vow101 and Vow1113 enzyme solutions were diluted to an activity of 10-15 U / mL with 0.1 mol / L Britton-Robinson buffer (pH 6.0). 1 mL of diluted enzyme solution was mixed thoroughly with 1 mL of gelatin substrate, and the enzyme activity was measured at 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃.

[0119] 3.3 Optimal pH

[0120] The activities of collagenases Vow101 and Vow1113 were determined at 40℃ and different pH conditions (pH 3-10). The method for determining the gelatin-degrading enzyme activity followed the procedure described above. Prepare 0.1 mol / L Britton-Robinson buffers with pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. Dilute the Vow101 and Vow1113 enzyme solutions with these buffers to an enzyme activity of 10-15 U / mL. The hydrolytic activity of collagenases on the substrate gelatin was then determined at 40℃ and the corresponding pH conditions.

[0121] Figure 2 The results of enzyme activity measurements under different temperatures (top) and pH conditions (bottom) are shown. The relative enzyme activity in each graph is obtained by dividing the enzyme activity at different temperatures or pH conditions by the enzyme activity at the optimum temperature or pH condition; that is, the enzyme activity at the optimum temperature or pH condition is defined as 100%. Figure 2 As shown in the upper figure, the optimal hydrolysis temperature for the wild-type enzyme Vow101 is 40℃, while the optimal hydrolysis temperature for the 12-site mutant enzyme Vow1113 is increased to 60℃, indicating improved heat resistance. Vow1113 is more suitable for the high-temperature conditions of fish skin collagen enzymatic hydrolysis in industrial practice. Figure 2 As shown in the lower figure, the pH adaptability of the mutant enzyme Vow1113 is consistent with that of the wild-type enzyme Vow101, with an optimal pH of 6.0 for both.

[0122] 4. Enzymatic preparation of CTP-rich collagen peptides

[0123] Pretreatment of Atlantic cod skin: Collect fresh Atlantic cod skin and rinse it thoroughly with running water to remove blood, mucus, scales, and other impurities. Remove any remaining fish meat, bones, and connective tissue from the skin. Then, cut the treated skin into 2-3 cm pieces. Place the skin pieces in an organic solvent (such as n-hexane) at a mass-to-volume ratio of 1 g fish skin to 5 mL n-hexane and stir and soak at room temperature for 2 hours. Hexane effectively dissolves the fat in the skin. After soaking, separate the skin from the hexane solution using filtration. Rinse the skin repeatedly with water. Then, add 3 times the volume of water (1 g skin to 3 mL water) to the defatted Atlantic cod skin, heat to 70-80°C, and maintain the temperature for 30 minutes to allow the gelatin in the skin to fully swell, yielding a crude collagen solution.

[0124] Enzymatic hydrolysis of fish skin collagen: Collagen peptides were prepared using a three-step enzymatic hydrolysis method. First, the skin was treated with alkaline protease KT124 enzyme solution (the preparation method of KT124 enzyme solution is described in patent CN119799680A). Next, it was treated with flavor protease Flavorzyme (purchased from Novozymes). Finally, it was treated with collagenase Vow1113. The specific steps are as follows:

[0125] (1) When the crude collagen solution is cooled to 50°C, add alkaline protease KT124 solution at 0.5‰ (w / w) of the dry weight of fish skin, adjust the pH of the reaction to 8.0-9.0 with sodium hydroxide solution, and stir the reaction at 50°C for 3 hours.

[0126] (2) Adjust the pH of the reaction mixture to 7.0-7.5 with acetic acid, add commercially available Novozymes flavor enzyme at 0.2‰ (w / w) of the dry weight of fish skin, and stir at 50℃ for 2 hours.

[0127] (3) Raise the temperature of the reaction mixture to 60°C, adjust the pH to 6.0, add the Vow1113 collagenase enzyme solution prepared in the above example at 0.4‰ (w / w) of the dry weight of the fish skin, and stir at 60°C for 3 h.

[0128] (4) Raise the temperature of the reaction mixture to 90°C and heat for 30 min to inactivate the enzyme and terminate the enzymatic hydrolysis. Centrifuge and take the middle layer liquid, which is the enzymatic hydrolysate of Atlantic cod skin collagen.

[0129] Decontamination and filtration of the enzymatic hydrolysate: Washed powdered activated carbon was added at a ratio of 0.5% (w / w) of the dry weight of the fish skin. The mixture was stirred to ensure uniform dispersion of the activated carbon in the enzymatic hydrolysate. The hydrolysate was subjected to an adsorption reaction at 40°C for 1 hour to remove pigments, odors, and other impurities. After the adsorption reaction, the mixture was centrifuged at 8000g for 20 minutes, and the supernatant was collected. A nanofiltration ceramic membrane with a retention cutoff of less than 5000 Da was used to filter and separate the enzymatic hydrolysate after adsorption of active peptides, yielding a clear fish skin collagen peptide solution rich in tripeptides.

[0130] Determination of peptide molecular weight distribution in enzymatic hydrolysate: The molecular weight and proportion of peptides in the enzymatic hydrolysate of Atlantic cod skin were determined by high-performance liquid chromatography (HPLC). Four standards were selected: aprotinin (Mr=6500 Da), bacitracin (Mr=1450 Da), tetrapeptide GGYR (Mr=451 Da), and tripeptide GGG (Mr=189 Da). A TSK gelG2000 SWXL column (7.8 × 300 mm) manufactured by Tosoh Corporation, Japan, was used as the mobile phase, with an aqueous solution containing 0.1% (v / v) trifluoroacetic acid (TFA) and 45% (v / v) acetonitrile as the mobile phase, at a flow rate of 0.5 mL / min, and at a wavelength of 220 nm. A standard curve was plotted using the peak elution time and molecular weight of each standard. The enzymatic hydrolysate prepared in the above examples was diluted to 10 mg / mL, filtered through a 0.22 µm membrane, and 20 µL was taken for sample determination. By comparing with standard peptide molecular weight, the molecular weight distribution of peptides and the proportion of peptides in the enzymatic hydrolysate prepared after the three-step enzymatic hydrolysis were calculated.

[0131] Calculation of the proportion of collagen tripeptides: Using the concentration of the tripeptide standard GGG as the abscissa (x, mg / mL) and the corresponding peak area as the ordinate (y), a linear regression was performed to obtain the standard curve equation and correlation coefficient (R² ≥ 0.999). The enzymatic hydrolysate sample was injected and measured under the above chromatographic conditions. The total area of ​​all chromatographic peaks within the integrated tripeptide retention time window was substituted into the standard curve equation, and combined with the sample dilution factor, the total amount of tripeptides in the enzymatic hydrolysate mixture (unit: mg / mL) was calculated. The calculation formula is as follows: Total tripeptide (mg / mL) = (Total peak area of ​​sample × concentration of standard × dilution factor) / peak area of ​​standard.

[0132] Glycine-proline-hydroxyproline (GPH) content determination: A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with a detection wavelength of 220 nm. The mobile phase consisted of phase A (0.1% (v / v) TFA in ultrapure water) and phase B (0.1% (v / v) TFA in acetonitrile solution), eluted isocratically at A:B = 95:5 (v / v), with a flow rate of 1.0 mL / min and a column temperature of 30 °C. The injection volume was 20 μL. A series of prepared GPH standard solutions were injected sequentially. Linear regression was performed with GPH concentration as the abscissa (x, μg / mL) and the corresponding peak area as the ordinate (y) to obtain the standard curve equation and correlation coefficient (R² ≥ 0.999). Inject the pretreated enzymatic hydrolysate sample and determine the concentration under the chromatographic conditions described above. Integrate the area of ​​the single chromatographic peak corresponding to GPH, substitute it into the standard curve equation, and calculate the GPH content (unit: μg / mL) in the enzymatic hydrolysate mixture based on the sample dilution factor. The calculation formula is as follows: GPH content (μg / mL) = (Sample GPH peak area × Standard concentration × Dilution factor) / Standard peak area.

[0133] The results (see Table 2) confirm that the peptide solution prepared by the three-step enzymatic hydrolysis method provided by this invention contains 80.17% peptides with a molecular weight less than 500 Da (<500 Da), significantly higher than the 60.32% of the wild-type enzyme Vow101. This high proportion of small molecular weight peptides is beneficial for maintaining the excellent bioactivity of fish skin collagen peptides. The proportion of collagen tripeptides is 36.53%, of which the GPH content reaches 3.14%. Figure 3 The HPLC chromatograms of GPH and standards in the enzymatic hydrolysate are shown.

[0134] Table 2. Molecular weight distribution and percentage of peptides in the enzymatic hydrolysate

[0135]

[0136] 5. Evaluation of the moisturizing properties of collagen peptides

[0137] The enzymatic hydrolysate of fish skin collagen prepared in the above examples was spray-dried to obtain collagen peptide powder rich in tripeptides. Following the method of Wang et al. (Wang J, Jin WH, Hou Y, et al. Chemical composition and moisture-absorption / retention ability of polysaccharides extracted from five algae [J]. International Journal of Biological Macromolecules, 2013, 57:26-29.), the moisturizing and hygroscopic properties of the collagen peptides were determined in vitro, with glycerin and hyaluronic acid, commonly used ingredients in cosmetics, serving as controls.

[0138] 5.1 Moisturizing properties

[0139] Collagen peptide powder, glycerin, and hyaluronic acid were pre-dried to constant weight. A saturated potassium carbonate solution was placed in a desiccator to create a dry environment with a relative humidity (RH) of 43%; a saturated ammonium sulfate solution was placed in a desiccator to create a humid environment with a RH of 81%. 1g of each of the 1% (w / w) aqueous solutions of collagen peptide powder, glycerin, and hyaluronic acid were prepared and placed in weighing bottles. The weighing bottles were placed in desiccators at 43% and 81% relative humidity, respectively. After 12h, 24h, 36h, 48h, and 60h, the weighing bottles were removed, weighed, and quickly returned. The humidification rate was calculated using the following formula: Humidification rate = m2 / m1 × 100%; where m1 is the initial mass of the sample (g); and m2 is the final mass of the sample after the specified time (g).

[0140] 5.2 Hygroscopicity

[0141] Weigh 1g of constant-weight collagen peptide powder, glycerin, and hyaluronic acid into weighing bottles. Place the weighing bottles in desiccators with relative humidity of 43% and 81%. After 12h, 24h, 36h, 48h, and 60h, remove the weighing bottles, weigh them, and quickly put them back. Calculate the moisture absorption rate using the following formula: Moisture absorption rate = (m4 - m3) / m3 × 100%; where m3 is the initial mass of the sample before moisture absorption (g); and m4 is the final mass of the sample after a specific moisture absorption time (g).

[0142] Figure 4The graph shows the trend of moisturizing rate over time under environmental conditions of relative humidity of 81% (top) and 43% (bottom). It can be observed that the moisturizing rate of all four samples gradually decreased with time, but the moisturizing performance of collagen peptides prepared by collagenases Vow101 and Vow113 was higher than that of glycerol and hyaluronic acid. In a humid environment with high humidity (RH 81%), after 60 hours of storage, the collagen peptides prepared by enzyme Vow1113 exhibited excellent moisturizing performance, with a moisturizing rate of 98.23%, higher than hyaluronic acid's 96.50% and glycerol's 95.34%. Even in a dry environment with a relative humidity of only 43%, after 60 hours of storage, the collagen peptides still maintained a high moisturizing rate of 94.57%, also exceeding hyaluronic acid's 92.31% and glycerol's 87.26%.

[0143] Figure 5 The trend of moisture absorption rate over time is shown under environmental conditions of 81% (top) and 43% (bottom) relative humidity, respectively. It can be observed that the moisture absorption rate of all four samples gradually increased over time and then tended to stabilize. The collagen peptides prepared by collagenases Vow101 and Vow113 exhibited higher moisture absorption properties than glycerol and hyaluronic acid. In a dry environment with 43% RH, the moisture absorption rate of all four samples began to stabilize after 24 hours. By 60 hours, the moisture absorption rate of the collagen peptides prepared by enzyme Vow1113 reached its maximum value of 19.25%, higher than 16.10% for glycerol and 9.74% for hyaluronic acid. In a humid environment with 81% RH, the moisture absorption rate of all four samples began to stabilize after 48 hours. After 60 hours, the hygroscopicity of collagen peptides prepared by enzyme Vow1113 reached a peak of 53.62%, which is higher than that of glycerol (44.39%) and hyaluronic acid (30.18%).

[0144] The foregoing results indicate that the collagen peptides prepared in this invention possess unique molecular structure advantages and biological activity. The collagen peptides prepared in this invention contain a high proportion of peptides with molecular weights below 500 Da and collagen tripeptides. These components contain more abundant hydrophilic groups, which can form hydrogen bonds with water molecules, thereby adsorbing and fixing water molecules around the collagen tripeptide molecules, exhibiting excellent hygroscopic properties. Furthermore, when combined with water molecules, they can form an effective protective structure on the surface, significantly inhibiting water evaporation, thus demonstrating outstanding moisturizing ability.

[0145] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A collagenase mutant, characterized in that, The amino acid sequence of the collagenase mutant is shown in SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the collagenase mutant as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

4. An expression vector or recombinant bacterial strain, characterized in that, The recombinant strain comprises the expression vector, which comprises the nucleic acid molecule as described in any one of claims 2-3.

5. An enzyme composition, characterized in that, This includes alkaline proteases, flavor proteases, and collagenase mutants as described in claim 1.

6. The use of the collagenase mutant as described in claim 1 or the enzyme composition as described in claim 5 in the preparation of collagen peptides.

7. A method for preparing collagen peptides, characterized in that, Includes the following steps: S1. Pre-treat the cleaned fish skin to obtain a crude collagen solution; S2. The crude collagen solution is sequentially subjected to alkaline protease, flavor protease and collagenase mutant as described in claim 1 for enzymatic hydrolysis, and then the enzymatic hydrolysis is terminated to obtain enzymatic hydrolysate, wherein the amount of alkaline protease, flavor protease and collagenase mutant is 0.1‰-0.5‰ of the dry weight of fish skin. S3. The enzymatic hydrolysate is subjected to deimpurity and deodorization treatment to obtain a purified collagen peptide solution.

8. The method for preparing collagen peptides according to claim 7, characterized in that, Step S2 specifically includes the following steps: S21. Add alkaline protease solution at 0.5‰ of the dry weight of fish skin, and stir and react for 3 hours at pH 8.0-9.0 and 50℃. S22. Add flavor protease at 0.2‰ of the dry weight of fish skin, and stir and react for 2 hours at pH 7.0-7.5 and 50℃. S23. Add collagenase mutant solution at 0.4‰ of the dry weight of fish skin, and stir and react for 3 hours at pH 6.0 and 60℃. S24. Raise the temperature of the reaction mixture to 90°C and heat for 30 min. Then centrifuge to collect the intermediate liquid to obtain the enzymatic hydrolysate.

9. The method for preparing collagen peptides according to claim 7, characterized in that, In step S1, the pretreatment includes degreasing and swelling.

10. The method for preparing collagen peptides according to claim 7, characterized in that, Step S3 specifically includes the following steps: Add activated carbon at 0.5% of the dry weight of fish skin, and perform adsorption reaction at 40℃ for 1 hour. Remove activated carbon by centrifugation, collect the supernatant, and filter it using a nanofiltration ceramic membrane with a retention cutoff of less than or equal to 5000 Da to obtain a purified collagen peptide solution.

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