Proline aminopeptidase mutant and application thereof in preparation of collagen peptide

Abstract

The invention belongs to the technical field of collagen peptide preparation, and particularly relates to a proline aminopeptidase mutant and application thereof in collagen peptide preparation. The amino acid sequence of the proline aminopeptidase mutant is as shown in SEQ ID NO. 2. The proline aminopeptidase mutant provided by the invention has the advantages of high enzyme activity, wide temperature and pH adaptation range, capability of adapting to a long-time high-temperature environment of collagen enzymolysis and the like, and is suitable for deep hydrolysis of collagen. Through cooperation with neutral protease, papain and collagenase, collagen raw materials from fish skin can be effectively degraded, the yield of low-molecular-weight peptide is increased, and collagen peptide with the molecular weight smaller than 1000 Da and accounting for 90% or above, collagen tripeptide accounting for 39% or above, collagen dipeptide accounting for 2% or above and the content of characteristic dipeptide Hyp-Gly being remarkably increased is generated. The method has important application value in the field of oriented preparation of collagen dipeptide.

Description

Technical Field

[0001] This invention relates to the field of collagen peptide preparation technology, and in particular to a proline aminopeptidase mutant and its application in collagen peptide preparation. Background Technology

[0002] Collagen peptides are low-molecular-weight bioactive peptides obtained through deep hydrolysis of collagen tissue. Among them, collagen dipeptides are the smallest and most easily absorbed active fragments after collagen hydrolysis. They consist of two amino acid residues, with typical sequences including Hyp-Gly and Pro-Hyp. They have core functions such as promoting the proliferation of skin fibroblasts, increasing hyaluronic acid synthesis, and improving skin moisture and elasticity. They have significant application value in functional foods / health products (such as anti-aging, skin care, and joint care oral liquids), biomedicine (such as wound dressings and tissue engineering scaffolds), and cosmetics (such as anti-wrinkle, moisturizing, and repairing serums, masks, and creams).

[0003] Currently, enzymatic hydrolysis is widely used in the preparation of collagen peptides due to its mild reaction conditions, ease of control, high specificity, and environmental friendliness. However, existing hydrolytic enzymes have significant technical bottlenecks: traditional proteases (such as papain and alkaline protease) have low substrate specificity and non-specific cleavage sites, making it difficult to efficiently break down the triple helix structure of fish skin collagen. This easily generates a large number of inactive heteropeptides, which in turn affects the yield of collagen dipeptides in collagen peptide products.

[0004] Prolyl aminopeptidases (PAPs, EC 3.4.11.5) are a class of exopeptidases widely found in bacteria, fungi, and plants and animals. Their core function is to specifically cleave proline (Pro) residues from the N-terminus of polypeptides, directionally hydrolyzing Pro-X peptide bonds (where X is any amino acid). Their unique hydrophobic pocket structure at the active site allows for precise recognition of peptides with a Pro N-terminus, achieving a specific hydrolysis rate exceeding 90% for characteristic sequences of fish skin collagen, making them ideal enzymes for the targeted preparation of collagen dipeptides. However, the preparation of collagen peptide products rich in dipeptides remains a significant challenge. Firstly, some currently isolated or commercially available prolyl aminopeptidases suffer from enzymatic instability and low catalytic efficiency, leading to incomplete collagen hydrolysis and a low content of small molecule peptides (<1000 Da) in the peptide distribution. Secondly, reaction conditions are limited. Most enzymes maintain activity only in neutral or weakly alkaline environments and have poor heat resistance (optimal temperature <50℃). Prolonged reactions at high temperatures will cause a sharp decrease in activity or complete inactivation. Therefore, the enzymatic hydrolysis process requires frequent pH and temperature adjustments, which greatly affects product distribution, yield stability, and significantly increases production costs. Given the specific advantages of PAPs and their potential in the targeted preparation of collagen dipeptides, there is an urgent need to develop novel PAPs or their variants with excellent properties, especially high temperature tolerance and / or high catalytic activity. This is of significant theoretical and practical importance for improving the yield of collagen-rich dipeptides and achieving their efficient and low-cost industrial production. Summary of the Invention

[0005] To address the problems of insufficient temperature tolerance and / or catalytic activity of proline aminopeptidase in existing technologies, and low dipeptide content in collagen hydrolysis products, this invention provides a novel proline aminopeptidase mutant with advantages such as high enzyme activity, wide temperature and pH adaptability, and the ability to adapt to the long-term high-temperature environment of collagen enzymatic hydrolysis, making it suitable for the deep hydrolysis of collagen. This invention also provides the encoding gene of the proline aminopeptidase mutant, an expression vector containing this encoding gene, and a recombinant strain, and further provides an enzyme composition containing this proline aminopeptidase 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 proline aminopeptidase mutant, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] A second aspect of the present invention provides a nucleic acid molecule that encodes a proline aminopeptidase 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 a neutral protease, papain, collagenase, and a proline aminopeptidase mutant as described above.

[0014] The sixth aspect of the present invention provides the use of the proline aminopeptidase 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. Neutral protease and papain are added to the crude collagen solution for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzymatic hydrolysis is terminated. Then, collagenase and the proline aminopeptidase mutant as described above are added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzymatic hydrolysis is terminated to obtain the enzymatic hydrolysate.

[0018] The amount of the neutral protease, the papain, the collagenase, and the proline aminopeptidase mutant is 0.1‰-0.5‰ of the dry weight of the fish skin.

[0019] S3. The enzymatic hydrolysate is decolorized, deodorized, and deimpurified, and then dried to obtain collagen peptide powder.

[0020] Furthermore, in step S1, the pretreatment includes degreasing and swelling treatment.

[0021] Furthermore, step S2 specifically includes the following steps:

[0022] S21. Based on the dry weight of fish skin, add 0.5‰ neutral protease and 0.3‰ papain, and enzymatically hydrolyze for 4 hours at 40℃, pH 7.5 and 300 r / min. Then heat to 95℃ and keep warm for 30 minutes to terminate the enzymatic hydrolysis.

[0023] S22. Based on the dry weight of fish skin, add 0.5‰ collagenase and 0.3‰ proline aminopeptidase mutant, and enzymatically hydrolyze for 2 hours at 60℃, pH 7.5 and 300 r / min. Then heat to 95℃ and keep warm for 30 minutes to terminate the enzymatic hydrolysis.

[0024] S23. Centrifuge the reaction mixture, collect the supernatant, and obtain the enzyme hydrolysate.

[0025] Further, step S3 specifically includes the following steps: add 1.0% powdered activated carbon based on the dry weight of fish skin, mix evenly, adsorb at 60℃ for 30 minutes, and after adsorption is completed, obtain collagen peptide powder by plate and frame filtration and spray drying.

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

[0027] The proline aminopeptidase mutant (P835) provided by this invention has advantages such as high enzyme activity, wide temperature and pH adaptability, and the ability to adapt to the long-term high-temperature environment of collagen hydrolysis, making it suitable for the deep hydrolysis of collagen. By combining with neutral proteases, papain, and collagenase, it can effectively degrade collagen raw materials derived from fish skin, improve the yield of low molecular weight peptides, and generate collagen peptides with a molecular weight <1000 Da content exceeding 90%, a collagen tripeptide content exceeding 39%, a collagen dipeptide content exceeding 2%, and a significantly increased content of the characteristic dipeptide Hyp-Gly. This has important application value in the targeted preparation of collagen dipeptides and is significant for improving the biological activity of collagen hydrolysates. 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 This is an SDS-PAGE electrophoresis image of the fermentation enzyme broth of the recombinant wild-type proline aminopeptidase P821 and its mutant P835 in the embodiments of the present invention.

[0030] Figure 2 This is a graph showing the optimal pH determination results for wild-type proline aminopeptidase P821 and its mutant P835 in the embodiments of the present invention.

[0031] Figure 3 This is a graph showing the optimal temperature determination results for wild-type proline aminopeptidase P821 and its mutant P835 in the embodiments of the present invention.

[0032] Figure 4This is a graph showing the thermostability test results of wild-type proline aminopeptidase P821 and its mutant P835 in an embodiment of the present invention.

[0033] Figure 5 The high-performance liquid chromatograms of GPH and standards in collagen peptide powder prepared by enzymatic hydrolysis of wild-type proline aminopeptidase P821 and its mutant P835 in this embodiment of the invention are shown.

[0034] Figure 6 The high-performance liquid chromatograms of Hyp-Gly and standards in collagen peptide powder prepared by enzymatic hydrolysis of wild-type proline aminopeptidase P821 and its mutant P835 are shown in this embodiment of the invention. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Currently, the enzymatic hydrolysis preparation process of collagen peptides has problems such as poor proline aminopeptidase activity, low hydrolysis efficiency, low content of low molecular weight peptides, especially low yield of dipeptides, which hinder the industrial production and application of collagen peptide products and collagen dipeptides.

[0048] To improve the activity and / or temperature tolerance of proline aminopeptidase, making it suitable for long-term high-temperature reactions of collagen hydrolysis and improving the hydrolysis efficiency of collagen, as well as increasing the yield of small molecular weight peptides, especially dipeptides, this invention mutated the key active site of proline aminopeptidase P821 (GenBank: AHK22714.1) from Aspergillus oryzae, and screened the target mutant P835 from a series of mutant enzymes. Compared to the wild-type P821, the mutant P835 has the following 11 site changes: amino acid at position 72 changes from G to A (G72A), amino acid at position 98 changes from D to N (D98N), amino acid at position 139 changes from E to Q (E139Q), amino acid at position 156 changes from W to F (W156F), amino acid at position 189 changes from P to A (P189A), amino acid at position 255 changes from F to Y (F255Y), amino acid at position 282 changes from T to S (T282S), amino acid at position 302 changes from H to Q (H302Q), amino acid at position 345 changes from D to E (D345E), amino acid at position 367 changes from K to R (K367R), and amino acid at position 392 changes from D to E (D392E).

[0049] The encoding genes of the wild-type proline aminopeptidase P821 and its mutant enzyme P835 were inserted into the pPIC9K expression vector and transformed into Pichia pastoris for extracellular secretion expression. The catalytic activity of the fermentation enzyme broths of each recombinant strain on L-proline-p-nitroaniline (L-Pro-pNA) substrates and their enzymatic hydrolysis ability on fish skin collagen were compared. The P835 mutant enzyme showed superior performance, exhibiting the following characteristics:

[0050] (1) The catalytic efficiency was significantly improved: When L-Pro-pNA was used as the substrate, the enzyme activity of the mutant enzyme P835 fermentation solution was measured to be 435 U / mL under the conditions of 55℃ and pH 7.5, which was 44% higher than that of wild type P821 (302 U / mL).

[0051] (2) Good environmental adaptability: The mutant enzyme P835 has a wide temperature and pH range; its optimal pH is 7.5, and it maintains more than 83% relative enzyme activity in the pH range of 7.0-9.0. Its optimal temperature is 65℃, and it maintains more than 75% relative enzyme activity in the range of 45-70℃.

[0052] (3) Improved temperature tolerance: After treatment at 50℃ for 2 h, the residual enzyme activity of mutant enzyme P835 was 82%, while that of wild enzyme P821 was 65%; after treatment at 55℃ for 2 h, the residual enzyme activity of mutant enzyme P835 was higher than 70%, while that of wild enzyme was only 40%; after treatment at 60℃ for 2 h, mutant enzyme P835 could still maintain more than 43% enzyme activity, while that of wild enzyme was less than 5%. It can be seen that mutant enzyme P835 has significantly enhanced heat resistance and thermal stability.

[0053] (4) Excellent collagen hydrolysis activity and characteristic peptide yield: After fish skin collagen was treated sequentially with a mixed enzyme solution of neutral protease and papain, and a mixed enzyme solution of collagenase and proline aminopeptidase of the present invention, the proportion of small molecular weight peptides with a molecular weight <1000 Da was 90.2%, the total amount of collagen tripeptides was 39.4%, the content of characteristic tripeptide Gly-Pro-Hyp was 3.2%, the total amount of collagen dipeptides was 2.4%, and the content of target dipeptide Hyp-Gly reached 1.6%, which were 10.9%, 26.7%, 14.3%, 118%, and 433% higher than that of wild type, respectively. It can be seen that the mutant enzyme P835 has significantly improved the deep hydrolysis activity of collagen, which is conducive to greatly increasing the content of low molecular weight peptides (<1000 Da) and tripeptides and dipeptides, especially the specific hydrolysis rate of oligopeptides with N-terminal Pro-X type peptide bonds is greatly improved compared with wild type.

[0054] Based on this, the present invention provides a proline aminopeptidase mutant, the amino acid sequence of which is shown below:

[0055] maaklvdkklhnvpgklrvaellfdvpvnycrpndgtlslfartvrrlttsfdapkgdkqlpwlvylqggpaygcrppqengwigtaldkgyqvlflnqrgtglcsnitagt lalkgnaikqaeylknfradnivrdcqavrrfltvdypedkrkfsiigqsfggfcavtylsmfptglaeaficgglaplvndpdpvyarnyekleernkayyskfpedierv krimqyleendvsvpsgrltparfqqlgliygmglsihelvlrawndleifgflthpsrtsidaaggfdgaviyailqesiycqgqasnwsadllrsananfcidaskpeiw ftgeminkemfdsydelneikeatdilattrdwpalndeaqlakyevpvyaatyiedmyahfshasssaakikgikqfitttmyhnalraksdevmqqlfalrddsid (see seq ID NO.2).

[0056] The proline aminopeptidase mutant (P835) provided by this invention has advantages such as high enzyme activity, wide temperature and pH adaptability, and the ability to adapt to the long-term high-temperature environment of collagen hydrolysis, making it suitable for the deep hydrolysis of collagen. By combining with neutral proteases, papain, and collagenase, it can effectively degrade collagen raw materials derived from fish skin, improve the yield of low molecular weight peptides, and generate collagen peptides with a molecular weight <1000 Da content exceeding 90%, a collagen tripeptide content exceeding 39%, a collagen dipeptide content exceeding 2%, and a significantly increased content of the characteristic dipeptide Hyp-Gly. This has important application value in the targeted preparation of collagen dipeptides and is significant for improving the biological activity of collagen hydrolysates.

[0057] Another embodiment of the present invention provides a nucleic acid molecule encoding a proline aminopeptidase mutant as described above.

[0058] The advantages of the nucleic acid molecule over the prior art are the same as those of the proline aminopeptidase mutant over the prior art as described above, and will not be repeated here.

[0059] 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 proline aminopeptidase 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.

[0060] For example, the gene sequence encoding a proline aminopeptidase mutant is shown in SEQ ID NO.4.

[0061] 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 proline aminopeptidase mutant of the present invention. Therefore, the sequences in the examples above should not be regarded as limiting the scope of protection of the present invention.

[0062] Another embodiment of the present invention provides an expression vector comprising the nucleic acid molecules described above.

[0063] The advantages of the expression vector over the prior art are the same as those of the proline aminopeptidase mutant over the prior art as described above, and will not be repeated here.

[0064] Optionally, the expression vector further includes gene sequences encoding a signal peptide, a promoter, and / or a terminator. The proline aminopeptidase 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 proline aminopeptidase mutant. That is, when all the aforementioned expression elements are included, the gene connection sequence from upstream to downstream is: promoter—signal peptide—proline aminopeptidase mutant—terminator. The signal peptide is used to guide the secretory expression of proline aminopeptidase 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 proline aminopeptidase 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.

[0065] 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.

[0066] 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.

[0067] Pichia pastoris (Komagataella phaffii), formerly known as Pichia pastoris, is a high-quality eukaryotic expression host widely used in the production of recombinant proteins. Its recombinant protein expression system has advantages such as high protein folding efficiency, strong secretory expression capacity, no endotoxin residue, ease of high-density fermentation, low host cell protein content, and simple downstream purification processes. It has been widely used in the industrial production of human therapeutic proteins, food, and feed enzymes. In a preferred embodiment of the present invention, a Pichia pastoris expression system is used, the expression vector is the Pichia pastoris expression vector pPIC9K, and the host (or recipient) cell is Pichia pastoris GS115. The pPIC9K vector contains the strong promoter P... aox1 (Methanol-induced) secreted signal peptide SP α-factor Histidine auxotype (His) - ) and the genetic mycotoxin resistance gene G418, terminator T aox1 When recombinantly expressing the proline aminopeptidase gene of this invention, the proline aminopeptidase gene sequence is placed between the pPIC9K multiple cloning sites to construct a recombinant expression vector suitable for secretory expression. After transforming the recombinant expression vector into Pichia pastoris, efficient secretory expression of proline aminopeptidase can be achieved through conventional fermentation culture.

[0068] 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.

[0069] 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.

[0070] Optionally, the recombinant strain originates from Pichia pastoris, specifically Pichia pastoris GS115.

[0071] 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.

[0072] This invention also provides a method for preparing a proline aminopeptidase mutant, comprising the following steps:

[0073] The recombinant strain described above was cultured, the fermentation broth was centrifuged, the supernatant was ultrafiltered, the concentrate was purified by anion exchange chromatography, the eluent was collected, and a purified enzyme solution containing the proline aminopeptidase mutant was obtained.

[0074] 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.

[0075] The present invention further provides an enzyme composition comprising the proline aminopeptidase mutant as described above. Specifically, the enzyme composition comprises a neutral protease, papain, collagenase, and the proline aminopeptidase mutant as described above.

[0076] The above-mentioned enzyme composition is combined for different enzyme cleavage sites. The various proteases are compounded and the collagen raw material from fish skin is enzymatically hydrolyzed in two steps. After decolorization and deodorization by activated carbon adsorption, impurity removal by plate and frame filtration, and spray drying, collagen peptide powder rich in small molecular weight peptides, especially rich in characteristic tripeptides and dipeptides, can be obtained.

[0077] Neutral protease and papain are commercially available. Collagenase is collagenase KT231, and its amino acid sequence and preparation method can be found in patent "CN119776327A A collagenase mutant, gene fragment, recombinant plasmid, recombinant expression system and its uses, and a method for preparing collagenase mutant and collagen tripeptide (publication date: 2025-04-08)".

[0078] The present invention also provides the application of the proline aminopeptidase mutant or its enzyme composition as described above in the preparation of collagen peptides, and provides related preparation methods.

[0079] The preparation method of collagen peptides in this embodiment of the invention includes the following steps:

[0080] S1. Pre-treat the cleaned fish skin to obtain a crude collagen solution;

[0081] S2. Neutral protease and papain are added to the crude collagen solution for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the hydrolysis is terminated. Then, collagenase and the proline aminopeptidase mutant as described above are added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the hydrolysis is terminated to obtain the enzymatic hydrolysate. The amount of the neutral protease, papain, collagenase and proline aminopeptidase mutant is 0.1‰-0.5‰ of the dry weight of the fish skin.

[0082] S3. The enzymatic hydrolysate is decolorized, deodorized, and deimpurified, and then dried to obtain collagen peptide powder.

[0083] Optionally, the pretreatment in step S1 includes degreasing and swelling treatment.

[0084] More specifically, the preprocessing steps are as follows:

[0085] Fish skin, after removing fish meat, bones, and connective tissue, is added to an organic solvent at a ratio of 1:5 (g:mL) and soaked at room temperature until the fat is dissolved. The fish skin is then separated by filtration. The defatted fish skin is added to water at a ratio of 1:3 (g:mL) and kept at 70-80℃ for 30 minutes to allow the gelatin in the fish skin to fully swell, resulting in a crude collagen solution.

[0086] Optionally, step S2 specifically includes the following steps:

[0087] S21. Based on the dry weight of fish skin, add 0.5‰ neutral protease and 0.3‰ papain, and enzymatically hydrolyze for 4 hours at 40℃, pH 7.5 and 300 r / min. Then heat to 95℃ and keep warm for 30 minutes to terminate the enzymatic hydrolysis.

[0088] S22. Based on the dry weight of fish skin, add 0.5‰ collagenase and 0.3‰ proline aminopeptidase mutant, and enzymatically hydrolyze for 2 hours at 60℃, pH 7.5 and 300 r / min. Then heat to 95℃ and keep warm for 30 minutes to terminate the enzymatic hydrolysis.

[0089] S23. Centrifuge the reaction mixture, collect the supernatant, and obtain the enzyme hydrolysate.

[0090] Optionally, step S3 specifically includes the following steps:

[0091] Add 1.0% powdered activated carbon based on the dry weight of fish skin, mix well, and adsorb at 60℃ for 30 minutes. After adsorption is complete, obtain collagen peptide powder by plate and frame filtration and spray drying.

[0092] 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:

[0093] 1) Biomaterials:

[0094] L-proline-p-nitroaniline (L-Pro-pNA, purchased from Nanjing Peptide Industry, catalog number R41168|108321-19-3), aprotinin (purchased from Merck, catalog number Y0001154), bacitracin (purchased from Shanghai Aladdin, catalog number B105485), tetrapeptide GGYR and tripeptide GGG (synthesized by Nanjing Peptide Industry Biotechnology Co., Ltd.), glycine-proline-hydroxyproline (GPH, synthesized by Nanjing Peptide Industry Biotechnology Co., Ltd.), hydroxyproline-glycine (Hyp-Gly, synthesized by Nanjing Peptide Industry Biotechnology Co., Ltd.), neutral protease (Neutrase 0.8L from Novozymes), papain (Shanghai Yuanye Company, catalog number S10011), expression vector pPIC9K (purchased from Invitrogen, catalog number V17520), TSK gel G2000 SWXL column and octadecylsilane-bonded silica column were all commercially available.

[0095] 2) Culture medium:

[0096] (a) MD solid medium: 13.4 g / L yeast nitrogen base (YNB), 20 g / L glucose, 0.4 mg / L biotin and 20 g / L agar powder;

[0097] (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).

[0098] (c) BMMY liquid medium: replace 10 g / L glycerol in (b) with 20 g / L methanol.

[0099] 1. Cloning of wild-type and mutant proline aminopeptidase genes

[0100] In this embodiment, site-directed mutagenesis was performed on key active sites of proline aminopeptidase P821 (GenBank: AHK22714.1) from Aspergillus oryzae to construct a gene library of PAP mutant enzymes. Pichia pastoris (P. pastoris) GS115 was used as the host cell to express the genes of these mutant enzymes. After inducing secretory expression of the target genes, the cell culture supernatant was collected as the fermentation enzyme solution. Using L-proline-p-nitroaniline (L-Pro-pNA) as a substrate, by comparing the enzyme activity and enzymatic properties of different PAP mutant fermentation enzyme solutions, a proline aminopeptidase mutant with improved enzyme activity and temperature tolerance was screened and named P835.

[0101] Compared to wild-type proline aminopeptidase P821, the proline aminopeptidase mutant P835 has the following 11 site changes: G72A / D98N / E139Q / W156F / P189A / F255Y / T282S / H302Q / D345E / K367R / D392E.

[0102] The amino acid sequence of wild-type proline aminopeptidase P821 is shown below:

[0103]

[0104] The amino acid sequence of the proline aminopeptidase mutant P835 is shown below:

[0105]

[0106] Based on the codon bias of Pichia pastoris, the nucleotide sequences encoding the above wild-type sequences and mutants were optimized. The coding genes of P821 and P835 (excluding stop codons) are shown in Table 1.

[0107] Table 1. Gene (DNA) sequences of embodiments of the present invention

[0108]

[0109] 2. Recombinant expression of proline aminopeptidase

[0110] The Pichia pastoris recombinant expression system was used, with the expression vector pPIC9K, which contains the strong promoter P. aox1 (Methanol-induced) secreted signal peptide SP α-factor Histidine auxotype (His) - ) and the genetic mycotoxin resistance gene G418, terminator T aox1 The nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 were inserted between the EcoRI and NotI restriction sites of the Pichia pastoris expression vector pPIC9K, respectively, and transformed into Escherichia coli DH5α to construct recombinant expression vectors pKT821 and pKT835. Positive clones were screened and sequenced to verify the successful construction of the vectors.

[0111] With Pichia pastoris GS115 (His - Mut + The host bacteria, *Pichia pastoris*, possess strong methanol utilization and high protein secretion efficiency. Using electroporation, the recombinant expression vectors pKT821 and pKT835, linearized with the restriction endonuclease SacI, were transformed into *P. pastoris* GS115. The GS115 resuscitation solution was plated on MD solid plates (histidine-free) and incubated at 30°C for 72 h. High-copy recombinant strains were screened using G418 gradient plates (0.5-4.0 mg / mL) to obtain the corresponding recombinant expression yeasts, named *Pichia pastoris* KT821 and *Pichia pastoris* KT835, respectively.

[0112] 3. Preparation of proline aminopeptidase enzyme solution and analysis of its enzymatic properties

[0113] Single colonies of recombinant strains KT821 and KT835 were picked and inoculated into 50 mL of BMGY liquid medium, and cultured at 30℃ and 220 r / min for 24 h until OD reached. 600 =2.0-3.0; then centrifuge at 8000 g for 10 min at 4℃, discard the supernatant, collect all cells in the seed culture and transfer to 50 mL BMMY liquid medium, continue culturing at 30℃ and 220 r / min for 72 h. At 24 h and 48 h of culture, methanol (v / v) of 2% of the total volume of the medium is added to induce the secretory expression of the target gene. At 72 h of culture, centrifuge at 10000 g for 10 min, collect the supernatant, which is the enzyme solution containing proline aminopeptidase P821 or P835.

[0114] The fermentation enzyme solution was directly loaded onto the sample for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results are shown below. Figure 1 In the figure, lane M represents the protein molecular weight standard, lane ck represents the supernatant of yeast GS115 carrying the expression vector pPIC9K, and lanes P821 and P835 represent the enzyme solutions of P821 and P835, respectively. The gel image shows the presence of a protein with a molecular weight of approximately 50 kDa in the fermentation supernatant, indicating that both the wild-type enzyme P821 and the mutant enzyme P835 were successfully secreted and expressed in Pichia pastoris, with the concentration of the target protein P835 being 0.41 mg / mL.

[0115] 3.1 Enzyme activity assay

[0116] Solution preparation: (1) 10 mmol / L L-Pro-pNA stock solution: Accurately weigh 21.92 mg L-Pro-pNA, dissolve in dimethyl sulfoxide (DMSO) and bring to a final volume of 10 mL, seal in the dark, and store frozen at -20°C. Thaw and mix at room temperature before use; (2) 0.5 mmol / L L-Pro-pNA working solution: Take 0.5 mL of 10 mmol / L stock solution and bring to a final volume of 10 mL with 50 mmol / L Tris-HCl buffer (pH 7.5). Prepare fresh and use immediately (use within 2 hours of preparation); (3) 1 mol / L glacial acetic acid stop solution: Measure 57.5 mL of glacial acetic acid and bring to a final volume of 1000 mL with deionized water. Store at room temperature; (4) p-nitroaniline (pNA) standard series solutions: Accurately weigh 10.96 mg pNA, dissolve in DMSO and bring to a final volume of 10 mL to obtain 5 mmol / L stock solution; take 0.02, 0.04, 0.08, 0.16, and 0.32 mL of stock solution respectively, and make up to 10 mL with Tris-HCl buffer to obtain a series of standard solutions of 10, 20, 40, 80, and 160 μmol / L. Prepare fresh solutions and measure the absorbance (OD405) of each concentration at a wavelength of 405 nm. Plot a standard curve and fit the regression equation (R²). 2 ≥0.995).

[0117] The proline aminopeptidase P821 and P835 enzyme solutions collected by centrifugation were diluted with Tris-HCl buffer (pH 7.5) to a suitable concentration (ensuring the absorbance value of the enzymatic reaction is in the range of 0.2-0.6), and kept on ice to avoid repeated freeze-thaw cycles. An appropriate amount of enzyme solution was inactivated by heating in a 90℃ water bath for 30 min, cooled to room temperature, and then diluted with Tris-HCl buffer to the same concentration as the sample enzyme solution as a blank control. In a 2 mL centrifuge tube, 890 μL of Tris-HCl buffer, 100 μL of 0.5 mmol / L L-Pro-pNA working solution, and 10 μL of enzyme solution (sample group) were added. The mixture was incubated at 55℃ for 10 min, and the reaction was terminated by adding 200 μL of glacial acetic acid. The absorbance at 405 nm was measured. This absorbance value reflects the concentration of pNA (p-nitroaniline) released after substrate hydrolysis during the reaction, and is used to calculate the extracellular fermentation broth enzyme activity (U / mL) of the recombinant bacteria. The procedures for the blank control group and the sample group were synchronized. Each sample was repeated in triplicate.

[0118] One enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol pNA per minute. Enzyme activity assays showed that, with L-Pro-pNA as the substrate, the mutant enzyme P835 had an activity of 435 U / mL, which was 44% higher than that of the wild-type P821 (302 U / mL).

[0119] 3.2 Enzymatic Properties Analysis

[0120] The enzyme solution collected by centrifugation was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The concentrate was purified by DEAE-Sepharose Fast Flow anion exchange chromatography. The anion exchange column was equilibrated with 20 mmol / L Tris-HCl (pH 7.5). After loading the concentrate, it was eluted with a linear gradient of 20-200 mmol / L Tris-HCl (pH 7.5) for 10-15 column volumes at a flow rate of 1-2 mL / min. The enzyme activity peak was collected. The active component was concentrated by ultrafiltration to obtain a pure enzyme solution with a purity ≥95%.

[0121] Optimal pH determination: Prepare 50 mmol / L Tris-HCl buffer solutions with pH values ​​of 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10. Dilute the pure enzyme solution to an appropriate concentration (so that the absorbance value of the enzymatic reaction is within the range of 0.2-0.6). Using L-Pro-pNA as a substrate, determine the enzyme activity at a temperature of 55℃ and the corresponding pH conditions.

[0122] Determination of optimal temperature: Dilute the pure enzyme solution to a suitable concentration (so that the absorbance value of the enzymatic hydrolysis reaction is in the range of 0.2-0.6), and under pH 7.5 conditions, use L-Pro-pNA as substrate to determine the enzyme activity at temperatures of 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃.

[0123] Thermal stability determination: Proline aminopeptidase was heat-treated at different temperatures (40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃) for 2 h. Then, the enzyme activity of each treatment group was measured at pH 7.5 and temperature 55℃ using L-Pro-pNA as substrate. The initial enzyme activity before the heat treatment at the seven temperature points of 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃ was taken as 100%.

[0124] Figure 2-3 The enzyme activity assays under different pH and temperature conditions are shown. The relative enzyme activity in each graph is obtained by dividing the enzyme activity at different temperatures or pH by the enzyme activity at the optimum temperature or pH, where the optimum temperature or pH is defined as 100%. The graphs show that the optimum pH for mutant enzyme P835 is 7.5, and it maintains over 83% relative activity within the pH range of 7.0-9.0, consistent with the overall trend of wild-type enzyme P821 in terms of pH adaptability. Regarding temperature adaptability, mutant P835 shows a significant improvement over wild-type enzyme P821. The optimum temperature for wild-type enzyme P821 is 60℃, while the optimum temperature for mutant P835 is increased to 65℃.

[0125] Stability test results (see) Figure 4 The results showed that after incubation at 50℃ for 2 hours, the residual enzyme activity of wild-type enzyme P821 was only 65%, while that of mutant P835 increased to 82%. After incubation at 55℃ for 2 hours, the residual enzyme activity of wild-type enzyme plummeted to 40%, while mutant P835 could still maintain a relative enzyme activity of more than 70%. Even under the harsh conditions of incubation at 60℃ for 2 hours, mutant P835 could still maintain an enzyme activity of more than 43%, while wild-type enzyme had less than 5%. It can be seen that the thermostability advantage of mutant enzyme is extremely significant.

[0126] 4. Enzymatic preparation of collagen peptides rich in dipeptides and tripeptides

[0127] Pretreatment of Atlantic cod skin: Collect 100g of fresh Atlantic cod skin, 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. Cut the treated skin into 1cm x 1cm pieces. Place the skin pieces in an organic solvent (such as n-hexane) at a mass-to-volume ratio of 1g skin to 5mL hexane. 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 (1g skin: 3mL water) to the defatted Atlantic cod skin and heat to 70-80℃ for 30 minutes to allow the gelatin in the skin to fully swell, yielding a crude collagen solution.

[0128] Collagen enzymatic hydrolysis: Collagen peptides were prepared using a two-step enzymatic hydrolysis method. First, the peptides were treated with a mixed enzyme solution of neutral protease and papain. Then, they were treated with a mixed enzyme solution of collagenase KT231 (CN119776327A) and proline aminopeptidase (P835 or P821). The specific steps are as follows:

[0129] (1) Based on the dry weight of fish skin, add 0.5‰ (w / w) of neutral protease and 0.3‰ (w / w) of papain, and enzymatically hydrolyze for 4 h at 40℃, pH 7.5 and 300 r / min. Then heat to 95℃ and keep warm for 30 min to inactivate the enzyme.

[0130] (2) After the reaction mixture is cooled to 60°C, add 0.5‰ (w / w) collagenase KT231 and 0.3‰ (w / w) proline aminopeptidase P835 or P821 based on the dry weight of fish skin. Enzymatically hydrolyze for 2 h at 60°C, pH 7.5 and 300 r / min, and then heat to 95°C and keep warm for 30 min to inactivate the enzyme.

[0131] (3) Centrifuge 10,000g of the enzyme hydrolysate for 20 minutes, take the supernatant, and obtain the enzyme hydrolysate.

[0132] Decolorization, deodorization, impurity removal, and drying of the enzymatic hydrolysate: Washed powdered activated carbon was added at a ratio of 1.0 (w / w) of the dry weight of the fish skin, mixed evenly, and adsorbed at 60℃ for 30 min to perform decolorization, deodorization, and impurity removal; after the adsorption reaction, the mixture was filtered through a plate and frame filter and spray-dried (inlet air temperature 180℃, outlet air temperature 80℃) to obtain fish skin collagen peptide powder rich in dipeptides and tripeptides.

[0133] The proportion of peptides with different molecular weights, the total amount of collagen tripeptides, and the contents of tripeptide Gly-Pro-Hyp (GPH) and dipeptide Hyp-Gly (HG) in peptide powder were determined by high performance liquid chromatography (HPLC).

[0134] Peptide molecular weight distribution determination: Accurately weigh an appropriate amount of peptide powder sample, dissolve it in the mobile phase to prepare a solution with a concentration of 1 mg / mL, filter it through a 0.22 μm filter membrane, and set aside for use. The HPLC column was a Tosoh TSKgel G2000 SWXL (7.8 mm × 300 mm); the mobile phase was a mixture of acetonitrile, water, and trifluoroacetic acid at a volume ratio of 45:55:0.1 (45:55:0.1, v / v / v); the flow rate was 0.5 mL / min; the detection wavelength was 220 nm; the column temperature was 30 ℃; and the injection volume was 20 μL. Four standards were selected, including: aprotinin (Mr = 6500 Da), bacitracin (Mr = 1450 Da), tetrapeptide GGYR (Mr = 451 Da), and tripeptide GGG (Mr = 189 Da). A standard curve was plotted using the logarithm of the molecular weight of the standard against the retention time. The corresponding molecular weight was calculated based on the retention time of each peak in the test solution. The relative percentage content of peptides with different molecular weights was calculated using the peak area normalization method.

[0135] Detection of collagen characteristic peptide content: The HPLC column was an octadecylsilane-bonded silica column (4.6 mm × 250 mm, 5 μm); mobile phase A was 0.1% (v / v) TFA in ultrapure water, and mobile phase B was 0.1% (v / v) TFA in acetonitrile solution; gradient elution was performed according to the specified gradient elution program: 0-10 min, 5-15% B; 10-25 min, 15-30% B; 25-30 min, 30-95% B; 30-35 min, 95% B; flow rate was 1.0 mL / min; detection wavelength was 214 nm; column temperature was 30℃; injection volume was 20 μL. Glycine-proline-hydroxyproline (Gly-Pro-Hyp, GPH) and hydroxyproline-glycine (Hyp-Gly) standards were prepared into a series of reference solutions of different concentrations. The solutions were then determined using the same method. A standard curve was plotted with peak area against concentration, and the contents of Gly-Pro-Hyp and Hyp-Gly in the test sample were calculated.

[0136] The analysis results showed that the enzymatic hydrolysis performance of mutant enzyme P835 was significantly improved compared with wild-type enzyme P821. In the collagen peptides prepared by wild-type enzyme P821, small molecular weight peptides with a molecular weight <1000 Da accounted for 81.3%, and the total amount of collagen tripeptides was 31.1%, of which the content of the characteristic tripeptide Gly-Pro-Hyp was 2.8%, and the total amount of collagen dipeptides was only 1.1%, of which the content of the target dipeptide Hyp-Gly was 0.3%. However, after being catalyzed by mutant enzyme P835, the proportion of small molecular weight peptides with a molecular weight <1000 Da increased to 90.2%, the total amount of collagen tripeptides increased to 39.4%, and the content of the characteristic tripeptide Gly-Pro-Hyp was 3.2%, which is 14.3% higher than that of wild type; the total amount of collagen dipeptides increased significantly to 2.4% (an increase of 118%), of which the content of the target dipeptide Hyp-Gly reached 1.6%, which is 5.3 times that of wild-type enzyme P821, and the advantages of targeted preparation of collagen dipeptides are extremely prominent. Figure 5-6 Chromatograms showing the determination of GPH and Hyp-Gly content in fish skin collagen peptide powder obtained by enzymatic hydrolysis of wild-type enzyme P821 and mutant enzyme P835 are presented respectively.

[0137] 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 proline aminopeptidase mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO.

2.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the proline aminopeptidase 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. The expression vector or recombinant strain according to claim 4, characterized in that, The expression vector is the Pichia pastoris expression vector pPIC9K, and the recombinant strain is Pichia pastoris GS115.

6. An enzyme composition, characterized in that, It includes neutral protease, papain, collagenase, and the proline aminopeptidase mutant as described in claim 1.

7. The use of the proline aminopeptidase mutant as described in claim 1 or the enzyme composition as described in claim 6 in the preparation of collagen peptides.

8. 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. Neutral protease and papain are added to the crude collagen solution for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzymatic hydrolysis is terminated. Then, collagenase and the proline aminopeptidase mutant as described in claim 1 are added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzymatic hydrolysis is terminated to obtain the enzymatic hydrolysate. The amount of the neutral protease, the papain, the collagenase, and the proline aminopeptidase mutant is 0.1‰-0.5‰ of the dry weight of the fish skin. S3. The enzymatic hydrolysate is decolorized, deodorized, and deimpurified, and then dried to obtain collagen peptide powder.

9. The method for preparing collagen peptides according to claim 8, characterized in that, Step S2 specifically includes the following steps: S21. Based on the dry weight of fish skin, add 0.5‰ neutral protease and 0.3‰ papain, and enzymatically hydrolyze for 4 hours at 40℃, pH 7.5 and 300r / min. Then heat to 95℃ and keep warm for 30 minutes to terminate the enzymatic hydrolysis. S22. Based on the dry weight of fish skin, add 0.5‰ collagenase and 0.3‰ proline aminopeptidase mutant, and enzymatically hydrolyze for 2 hours at 60℃, pH 7.5 and 300 r / min. Then heat to 95℃ and keep warm for 30 minutes to terminate the enzymatic hydrolysis. S23. Centrifuge the reaction mixture, collect the supernatant, and obtain the enzyme hydrolysate.

10. The method for preparing collagen peptides according to claim 8, characterized in that, Step S3 specifically includes the following steps: Add 1.0% powdered activated carbon based on the dry weight of fish skin, mix well, and adsorb at 60℃ for 30 minutes. After adsorption is complete, filter through a plate and frame filter and spray dry to obtain collagen peptide powder.

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