阅读说明：本技术 胶原蛋白水解酶及其编码基因、制备方法和用途 (Collagen hydrolase and its coding gene, preparation method and use ) 是由 郭玉杰 张春晖 李侠 张鸿儒 于 2021-11-03 设计创作，主要内容包括：本发明公开了一种胶原蛋白水解酶,其氨基酸序列如SEQ ID NO.1所示。本发明还公开了胶原蛋白水解酶基因、胶原蛋白水解酶基因重组载体、胶原蛋白水解酶基因工程菌、胶原蛋白水解酶的制备方法和胶原蛋白水解酶的用途。本发明的胶原蛋白水解酶具有能够降解骨胶原蛋白,并提高小分子量骨胶原蛋白肽的得率。(The invention discloses a collagen hydrolase, and the amino acid sequence of the collagen hydrolase is shown as SEQ ID NO. 1. The invention also discloses a collagen hydrolase gene, a collagen hydrolase gene recombinant vector, a collagen hydrolase gene engineering bacterium, a preparation method of the collagen hydrolase and application of the collagen hydrolase. The collagen hydrolase of the invention can degrade the collagen and improve the yield of the small molecular weight collagen peptide.)

1. The collagen hydrolase is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. A collagen hydrolase gene encoding the collagen hydrolase according to claim 1.

3. The collagen hydrolase gene according to claim 2, wherein the DNA sequence is represented by SEQ ID No.2 and the cDNA sequence is represented by SEQ ID No. 4.

4. A recombinant vector for a collagen hydrolase gene, comprising the collagen hydrolase gene according to claim 2.

5. A collagen hydrolase gene-engineered bacterium comprising the collagen hydrolase gene recombinant vector according to claim 4.

6. The method for preparing collagen hydrolase according to claim 1, comprising the steps of:

s1 extraction of host strainRhizopusoryzaeThe RNA sequence of CGMCC3.17463, obtaining the cDNA sequence of a host strain through reverse transcription, carrying out PCR amplification by taking the cDNA sequence of the host strain as a template to obtain the cDNA sequence of collagen hydrolase, and then connecting the cDNA sequence of the collagen hydrolase to a pichia pastoris expression vector pPIC9 to obtain a collagen hydrolase gene recombinant vector;

s2, transforming the collagen hydrolase gene recombinant vector into a pichia pastoris Gs115 host cell to obtain a collagen hydrolase gene engineering strain;

s3, activating the collagen hydrolase gene engineering strain, culturing for 2-3d at 30 ℃, and expressing the collagen hydrolase gene engineering strain under the induction of methanol to produce a crude enzyme solution of the collagen hydrolase;

s4, concentrating and purifying the crude enzyme solution to obtain the purified collagen hydrolase.

7. The method of claim 6, wherein the step S1 of performing PCR amplification using the cDNA sequence of the host strain as a template to obtain the cDNA sequence of the collagen hydrolase comprises the steps of:

s1a, designing and synthesizing specific primer pair of collagen proteolytic enzymeRoAPA _ F andRoAPA _ R, wherein, RoAPA_Fthe sequence of (A) is shown in SEQ ID NO.5, RoAPA_Rthe sequence of (A) is shown in SEQ ID NO. 6;

s1b, using cDNA of host strain obtained by reverse transcription as template, andRoAPA _ F andRoand (3) carrying out PCR amplification by using APA _ R as a primer to obtain a cDNA sequence of the collagen hydrolase.

8. The method of producing a collagen hydrolase according to claim 7, wherein the conditions for obtaining the cDNA sequence of the collagen hydrolase by performing PCR amplification using the cDNA sequence of the host strain as a template in step S1 and step S1 are as follows: 3 min at 97 ℃; 30 s at 95 ℃, 30 s at 63 ℃ and 1 min at 72 ℃ for 32 cycles; 10 min at 72 ℃.

9. Use of a collagen hydrolyzing enzyme according to claim 1 for degrading collagen or for preparing a small molecular weight collagen peptide.

Technical Field

The invention relates to the field of biotechnology. More specifically, the invention relates to a collagen hydrolase, a coding gene, a preparation method and an application thereof.

Background

The livestock and poultry bones contain abundant ossein, which is an important raw material for preparing functional ossein peptide and has great utilization value. The small molecular weight ossein peptide prepared by enzymolysis of livestock and poultry ossein has the activities of resisting oxidation, resisting aging, promoting mineral absorption, resisting osteoporosis and the like. Meanwhile, the small molecular weight ossein protein peptide has the characteristics of good solubility, easy absorption and the like, and the absorption effect is better than that of collagen protein. The protease hydrolysis method is the most common method for preparing the small molecular weight ossein protein peptide at present, and has the advantages of low cost, simple operation, mild conditions and the like. However, due to the unique (G-X-Y) n repeating unit sequence and compact triple-helix structure of collagen, only a few proteases have the capability of catalyzing the hydrolysis of bone collagen, and few collagenases and production strains are commercially available at present. It has been shown by some studies that,Clostridium histolyticum、Bacillussp.，Candida albicansandVibrio vulnificusthe strains have the potential of generating collagen hydrolase, but most of the strains are pathogenic bacteria, and the pathogenic bacteria also generate corresponding toxin when the collagen hydrolase is generated, so that the strains are not suitable for preparing bone-derived food.

Disclosure of Invention

An object of the present invention is to provide a collagen hydrolase which can degrade collagen and increase the yield of small molecular weight collagen peptides, and a gene encoding the collagen hydrolase, a method for preparing the collagen hydrolase, and uses of the collagen hydrolase.

To achieve these objects and other advantages in accordance with the present invention, there is provided a collagen hydrolase having an amino acid sequence shown in SEQ ID NO. 1.

The invention also provides a collagen hydrolase gene which codes the collagen hydrolase.

Preferably, the DNA sequence of the collagen hydrolase gene is shown as SEQ ID NO.2, and the cDNA sequence is shown as SEQ ID NO. 4.

The invention also provides a collagen hydrolase gene recombinant vector, which comprises the collagen hydrolase gene.

The invention also provides a collagen hydrolase gene engineering bacterium which comprises the collagen hydrolase gene recombinant vector.

The invention also provides a preparation method of the collagen hydrolase, which comprises the following steps:

s1 extraction of host strainRhizopusoryzaeThe RNA sequence of CGMCC3.17463, obtaining the cDNA sequence of a host strain through reverse transcription, carrying out PCR amplification by taking the cDNA sequence of the host strain as a template to obtain the cDNA sequence of collagen hydrolase, and then connecting the cDNA sequence of the collagen hydrolase to a pichia pastoris expression vector pPIC9 to obtain a collagen hydrolase gene recombinant vector;

s2, transforming the collagen hydrolase gene recombinant vector into a pichia pastoris Gs115 host cell to obtain a collagen hydrolase gene engineering strain;

s3, activating the collagen hydrolase gene engineering strain, culturing for 2-3d at 30 ℃, and expressing the collagen hydrolase gene engineering strain under the induction of methanol to produce a crude enzyme solution of the collagen hydrolase;

s4, concentrating and purifying the crude enzyme solution to obtain the purified collagen hydrolase.

Preferably, in the method for preparing collagen hydrolase, in step S1, PCR amplification is performed using the cDNA sequence of the host strain as a template to obtain the cDNA sequence of collagen hydrolase, which specifically includes the following steps:

s1a, designing and synthesizing specific primer pair of collagen proteolytic enzymeRoAPA _ F andRoAPA _ R, wherein,RoAPA_Fthe sequence of (A) is shown in SEQ ID NO.5, RoAPA_Rthe sequence of (A) is shown in SEQ ID NO. 6;

s1b, using cDNA of host strain obtained by reverse transcription as template, andRoAPA _ F andRoand (3) carrying out PCR amplification by using APA _ R as a primer to obtain a cDNA sequence of the collagen hydrolase.

Preferably, in the method for preparing a collagen hydrolase, in step S1 and step S1, the cDNA sequence of the host strain is subjected to PCR amplification using the cDNA sequence as a template under the following conditions: 3 min at 97 ℃; 30 s at 95 ℃, 30 s at 63 ℃ and 1 min at 72 ℃ for 32 cycles; 10 min at 72 ℃.

The invention also provides an application of the collagen hydrolase, which is used for degrading collagen or preparing small molecular weight collagen peptide.

The invention at least comprises the following beneficial effects:

the invention provides a novel collagen hydrolase with collagen hydrolytic activity, and introduces the coding gene into an engineering strain by a bio-enzyme engineering method, so as to realize the heterologous expression of the collagen hydrolase and promote the industrial production of the collagen hydrolase.

Secondly, in the process of preparing the collagen peptide by the enzyme method, the collagen hydrolase provided by the invention can improve the yield of the small molecular weight collagen peptide.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 shows one embodiment of the collagen hydrolase of the present inventionRoThe relative enzyme activity curve chart of APA under different pH conditions;

FIG. 2 shows one embodiment of the collagen hydrolase of the present inventionRopH stability profile of APA;

FIG. 3 shows a collagen hydrolase according to one embodiment of the present inventionRoRelative enzyme activity curve chart of APA under different temperatures;

FIG. 4 shows a collagen hydrolase according to one embodiment of the present inventionRoTemperature stability profile of APA;

FIG. 5 shows a pair of different inhibitors according to one embodiment of the present inventionRoThe effect of APA catalytic activity;

FIG. 6 shows one of the solutions of the present inventionRoSDS-PAGE analysis of APA hydrolyzed collagen.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Test materials and reagents

1. Bacterial strain and carrier: the host strain is rhizopus oryzae (A)Rhizopusoryzae) Can be obtained from China general microbiological culture Collection center (CGMCC) with the number of CGMCC 3.17463; the engineering strain for heterologous expression of protein is Pichia pastoris (A)Pichia pastorisGS115) purchased from bio-engineering (shanghai) gmbh, and pichia pastoris expression vector pPIC9 purchased from Invitrogen corporation;

2. enzymes and other biochemical reagents: the endonuclease was purchased from TaKaRa, the ligase was purchased from Invitrogen, and the others were made by domestic reagents (all available from Biochemical reagents);

3. e.coli culture medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.000049< Biotin, 1% glycerol (v/v);

4. BMGY medium; 1% yeast extract, 2% peptone, 1.34% YNB, 0.000049< Biotin, 1% glycerol (v/v).

5. BMMY medium: the components were identical to BMGY except that 0.5% methanol was used instead of glycerin, and the pH was 4.0.

Note: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

Examples

1. Collagen hydrolaseRoObtaining the DNA sequence of APA

Extraction of host strainsRhizopusoryzaeThe DNA sequence of CGMCC3.17463 is preserved at-20 deg.c. Designed for collagen hydrolasesRoSpecific primer for cloning APA geneRoAPA _ F andRoAPA _ R, primerRoAPA _ F andRothe sequences of APA _ R are SEQ ID NO.5 and SEQ ID NO.6 respectively, and the host strainRhizopusoryzaeCarrying out PCR amplification by using a DNA sequence of CGMCC3.17463 as a template, wherein the amplification conditions are as follows: 3 min at 97 ℃; 30 s at 95 ℃, 30 s at 63 ℃ and 1 min at 72 ℃ for 32 cycles; 10 min at 72 ℃. Obtaining a DNA sequence of about 1252 bp, recovering the DNA sequence, and sequencing by Meiji biological medicine science and technology Limited company in Shanghai, wherein the gene sequence is shown in SEQ ID NO.2 and is collagen hydrolaseRoThe DNA sequence of APA, the corresponding amino acid sequence is SEQ ID NO. 1.

2. Collagen hydrolaseRoObtaining cDNA sequence of APA

Extraction of host strainsRhizopusoryzaeThe RNA sequence of CGMCC3.17463 is obtained by reverse transcription to obtain host strainRhizopusoryzaeThe cDNA sequence of CGMCC 3.17463. Design of cloning primersRoAPA _ F andRoAPA _ R, primerRoAPA _ F andRothe sequences of APA _ R are SEQ ID NO.5 and SEQ ID NO.6 respectively, and the host strainRhizopusoryzaeCarrying out PCR amplification by using the cDNA sequence of CGMCC3.17463 as a template to obtain collagen hydrolaseRoAnd (3) the cDNA sequence of the APA is obtained by the amplification, and the sequence obtained by the amplification is sent to the Shanghai Meiji Biotech limited company for sequencing, the length is 1194 bp, and the gene sequence is shown in SEQ ID NO. 4.

Analysis of collagen HydrolaseRoDNA sequence of APA and collagen hydrolaseRocDNA sequence information of APA, collagen hydrolaseRoThe total length of the DNA sequence of the APA is 1252 bp, the length of the DNA sequence is 58bp, and the base sequence is shown as SEQ ID NO. 3. Collagen hydrolaseRoThe deduced amino acid sequence of cDNA sequence of APA is predicted by software to find that 21 amino acids at N end are signal peptide sequence of protein. The collagen hydrolase is found by Blast comparisonRoThe highest similarity of the APA protein sequence and protease sequences published in a database is only 78.6%, the highest similarity of the APA protein sequence and related enzymes reported by the existing crystal structures is only 51.3%, and the results show that the APA protein sequence is derived from host strainsRhizopusoryzaeThe protease coding gene obtained by separation and cloning in CGMCC3.17463 has high novelty.

3. Preparation of recombinant engineered strains

(1) Preparation of recombinant engineered strains

To sequence the correct collagen hydrolaseRoThe cDNA of APA is used as a template, and primers with restriction sites of EcoR I and Not I are designed and synthesizedRoAPA _ F andRoAPA _ R, primerRoAPA _ F andRothe sequences of the APA _ R are SEQ ID NO.5 and SEQ ID NO.6 respectively, wherein, the primerRoAPA _ F sequence (CGGAATTCATGAAATTCACTCTTGTCTCTT) underlined are EcoR I restriction sites, primersRoAPA _ R sequence (TTGCGGCCGCTTATTTGTTTTGGTCAACAGAAGC) is Not I restriction site. With collagen hydrolaseRocDNA of APA as a template, toRoAPA _ F andRoAPA _ R is used as a primer to carry out PCR amplification, and then EcoR I and Not I are used for enzyme digestion of PCR products to obtain the amplified collagen hydrolaseRocDNA sequence of APA, collagen hydrolase after amplificationRoThe cDNA sequence of the APA is connected to a Pichia pastoris expression vector pPIC9 to obtain a recombinant expression vector pPIC9-RoAnd (4) APA. Namely collagen hydrolaseRoThe cDNA sequence of APA is inserted into the downstream of the signal peptide sequence of the expression vector to form a correct reading frame with the signal peptide to construct the Pichia pastoris expression vector pPIC9-RoAPA, then transformed into E.coli competent cells Trans1 in E.coli culture medium. And (4) carrying out DNA sequencing on the positive transformants, and using the transformants with correct sequencing to prepare a large amount of recombinant plasmids. The plasmid vector DNA sequence is linearly expressed by using restriction endonuclease Bgl II, pichia pastoris GS115 competent cells are transformed by electric shock, the cells are cultured for 2 to 3 days at the temperature of 30 ℃, transformants growing on an MD plate are selected for further expression experiments, and the specific operation refers to a pichia pastoris expression operation manual. And collagen-containing proteolytic enzymes were constructed in the same mannerRoExpression vector of cDNA sequence of APA signal peptide sequence and conversion.

(2) Screening of transformants having high collagenase Activity

A plurality of single colonies were picked from the MD plate with the transformant by using a sterilized toothpick, and spotted on another MD plate according to the number, and the MD plate was cultured in an incubator at 30 ℃ for 1 to 2 days until colonies grew out. Sequentially selecting transformants from the MD plate according to the numbers, respectively and correspondingly inoculating the transformants into centrifuge tubes filled with 3 mL of BMGY medium, and performing shake culture for 48 hours at the temperature of 30 ℃ and the rotation speed of 220 rpm; centrifuging the bacterial liquid cultured by a shaker for 48 h at 3000 Xg for 15 min, removing supernatant, adding 1 mL of BMMY culture medium containing 0.5% methanol into the centrifuge tube, and performing induction culture at 30 ℃ and 220 rpm; after the induction culture is carried out for 48 h, centrifuging for 5 min at 3000 Xg, taking the supernatant for enzyme activity detection, and screening out a transformant with high collagen hydrolase activity from the supernatant, wherein the concrete operation refers to a pichia pastoris expression operation manual.

4. Recombinant collagen hydrolaseRoPreparation of APA

(1) Recombinant engineered strain pPIC9-RoExpression of APA

Screening out transformants with higher enzyme activity, inoculating the transformants into 300 mL BMGY liquid medium, and carrying out shake culture on a shaker for 48 hours at the temperature of 30 ℃ and the rotating speed of 220 rpm; shaking culturing, centrifuging at 5000 rpm for 5 min, removing supernatant, adding 100 mL BMMY liquid culture medium containing 0.5% methanol into thallus, and inducing at 30 deg.C and 220 rpm for 72 h. During the induction culture period, the methanol solution is replenished once at intervals of 24 hours to compensate the loss of methanol, so that the concentration of the methanol is kept at about 0.5 percent; after induction culture for 72 h, centrifuging at 12000 Xg for 10 min, collecting supernatant fermentation liquor, detecting enzyme activity and carrying out SDS-PAGE protein electrophoresis analysis.

(2) Purifying to obtain recombinant collagen hydrolaseRoAPA

The supernatant of the recombinant engineered strain collagenase expressed in the shake flask was collected, concentrated by passing through a 10 kDa membrane, while the medium was replaced with a low salt buffer, and further concentrated by using a 10 kDa ultrafiltration tube. Concentrating the recombinant collagen hydrolase which can be diluted to a certain multipleRoAPA, and purifying by ion exchange chromatography to obtain recombinant collagen hydrolaseRoAnd (4) APA. Specifically, recombinant collagen hydrolase is takenRo2.0 mL of the APA concentrate was passed through a HiTrap Q Sepharose XL anion column equilibrated with 20 mM Tris-HCl (pH 7.5) in advance, followed by linear gradient elution with 0.1 mol/L NaClAnd detecting the enzyme activity and determining the protein concentration of the eluate collected in steps.

5. For collagen hydrolaseRoPartial characterization of APA

The collagen hydrolase prepared by the invention is developed by adopting a forskolin phenol reagent developing methodRoAPA was assayed for activity. The specific method comprises the following steps: hydrolyzing collagen protein with enzymeRoAfter APA reacts with 1 mL of reaction system for 10 min, 1 mL of trichloroacetic acid (0.4 mol/L) is added to stop the reaction, wherein the pH of the 1 mL of reaction system is 3.0, the temperature is 30 ℃, and the reaction system contains 500 muL of appropriate diluted enzyme solution and 500 muL of substrate; after the reaction is terminated, the reaction system is centrifuged at 12000 rpm for 3 min, 2.5 mL of sodium carbonate (0.4 mol/L) is added into 500 muL of supernatant, 500 muL of forlin phenol reagent is added, color development is carried out for 20 min at the temperature of 40 ℃, and the OD value is measured under the condition of ultraviolet wavelength of 680 nm after cooling. Protease activity unit definition: under certain conditions, the amount of enzyme required to decompose substrate casein to generate l μmol tyrosine per minute is 1 activity unit (U).

(1) Collagen hydrolaseRoDetection of optimum pH and pH stability of APA

The collagen hydrolase obtained by purification of the inventionRoAPA was subjected to enzymatic reactions at different pH conditions to determine its optimum pH. The buffer solution comprises glycine-hydrochloric acid buffer solution with pH of 2.0-3.0, citric acid-disodium hydrogen phosphate buffer solution with pH of 3.0-8.0, and Tris-HCl buffer solution with pH of 8.0-10.0. Purifying the obtained collagen hydrolaseRoThe results of the determination of the optimum pH of APA in a buffer system with different pH values at a temperature of 55 ℃ are shown in FIG. 1: collagen hydrolase at 55 deg.CRoThe optimum pH value of APA is 3.0, and the enzyme can maintain higher enzyme activity within the pH range of 3.0-4.0.

The enzyme solution was treated in buffers of different pH values at 30 ℃ for 60 min, and the enzyme activity was measured to investigate the pH stability of the enzyme. The results are shown in FIG. 2 and show that: collagen hydrolaseRoThe pH value of APA is 3.0-6.0, more than 90% of enzyme activity can be maintained, and the enzyme has good pH stability under acidic conditions.

(2) Collagen hydrolaseRoDetection of APA optimum reaction temperature and thermal stability

Purified collagen hydrolaseRoAPA was assayed for enzyme activity at different temperatures (30-70 ℃) at pH 3.0, as shown in FIG. 3: the optimal reaction temperature of the enzyme is 55 ℃, and the enzyme still has more than 80% of enzyme activity at 60 ℃. Purified collagen hydrolaseRoAPA is treated at 50 deg.C, 55 deg.C and 60 deg.C for different time respectively, and then enzyme activity is measured at 55 deg.C. The results are shown in FIG. 4, which shows collagen hydrolaseRoThe APA treatment at 60 deg.C for 30 min can completely inactivate protein. In conclusion, the collagen hydrolase has been shown to beRoThe APA has high-efficiency proteolytic activity at 50-60 ℃, and the protease can be completely inactivated after incubation for 30 min at 60 ℃. This means that the protease of the present invention has an important application value in the fields of food, medicine, and the like.

(3) Collagen hydrolase with different metal ion/reagent pairsRoEffect of APA Activity

To determine the different metal ion pairs of collagen hydrolasesRoEffect of APA Activity in determining collagen hydrolaseRoBefore the catalytic activity of APA, Mn is added into the reaction system²⁺、Cu²⁺、Cr³⁺、Zn²⁺、K⁺、Na⁺、Ca²⁺、Mg²⁺、Ni²⁺、Co^{2 +}、Pb²⁺、Fe³⁺Controlling the final concentration of different metal ions to be 3 mM, and adjusting the pH value of the solution to be 3.0; the protease activity assay was then performed at 55 ℃. The results show that Mn²⁺And Cu²⁺For collagen hydrolaseRoThe APA has obvious activating effect, Pb²⁺And Fe³⁺For collagen hydrolaseRoThe APA proteolytic activity has obvious inhibiting effect, and other metal ions have collagen hydrolase activityRoThe APA activity had essentially no effect. At the same time, we analyzed the addition of different types of protease inhibitors (blank CK, 2 mM cystatin E-64, 2 mM serpin PMSF, 5 mM metalloprotease inhibitor EDTA,and 0.05 mM aspartic protease inhibitor pepstatin A) on collagen hydrolaseRoThe effect of the activity of APA is shown in figure 5. The research shows that the collagen hydrolaseRoThe specific inhibition of APA by Peptatin A, Peptatin A can be specifically combined with the catalytic pocket of aspartic protease but not be cut, thereby inhibiting the activity of catalytic residue, and further proving that the collagen hydrolaseRoAPA belongs to the aspartic protease family.

6. Collagen hydrolaseRoApplication of APA in preparation of small molecular weight ossein protein peptide

(1) Enzymolysis of ossein protein

Accurately weighing 5.0 g of collagen, and adding 1000 mL of water to prepare 0.5% (w/v) collagen solution; adjusting pH of the solution to 3.5, adding collagen hydrolase in an amount of 5000U/gRoAPA, stirring, performing enzymolysis at 50 deg.C, heating in boiling water bath for 10 min to allow collagen hydrolaseRoThe APA was completely inactivated. Mixing 0.25 mL ossein enzymolysis solution with equal volume of 10% TCA, shaking up with shaking to 10000gAnd centrifuged at 4 ℃ for 20 min. And (3) sucking 50 mu L of the supernatant, sequentially adding the supernatant into a 96-well plate, sequentially adding 200 mu L of BCA working solution, and incubating for 2 h at room temperature. The resulting mixture was placed in a microplate reader, and absorbance was measured at a wavelength of 562 nm. Researches show that the hydrolysis degree gradually increases along with the extension of the enzymolysis time, and the hydrolysis degree does not increase along with the extension of the time when the plateau period is reached after about 4 hours. The above results indicate that the collagen hydrolaseRoAPA can catalyze the hydrolysis of bone collagen under acidic condition. The collagen hydrolysate was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE), and the results showed that collagen was hydrolyzed by collagen hydrolaseRoAPA hydrolysis, obvious molecular weight reduction, characteristic band (alpha) of ossein protein₁And alpha₂Bands) gradually disappeared as the reaction time was extended (fig. 6). The above results indicate that the collagen hydrolaseRoThe APA can catalyze the hydrolysis of the ossein protein to generate small molecular peptides, and has application potential in the industrial preparation of the ossein protein peptides.

(2) Determination of molecular weight distribution of collagen enzymatic hydrolysate

The molecular weight distribution of the collagen hydrolysate was determined using an Agilent HPLC1260-II system (Agilent Technologies Inc., California, USA). TSK gel G2000 SWXL chromatography column (7.8X 300 mm, TOSOH, Tokyo, Japan); column temperature: 40 ℃; mobile phase: a is a 45% (v/v) acetonitrile solution mixed with 0.1% trifluoroacetic acid; performing equal gradient elution; flow rate: 0.5 mL/min; sample introduction volume: 10 μ L, and the response value was measured at a wavelength of 214 nm. A standard curve (Y = -3.9331X +27.517, R2= 0.987) between retention time (X) and logarithm of molecular weight (Y) is established by taking Gly-Sar (146 Da), Gly-Gly-Tyr-Arg (451 Da), Bacitracin (1422 Da), Aprotinin (6511 Da) and Cytochrome C (12327 Da) as standards. Table 1 shows collagen hydrolasesRoThe molecular weight distribution of collagen peptide prepared by hydrolyzing collagen with APA was found to be comparable to pepsin and trypsinRoThe collagen peptide obtained by APA hydrolysis has more advantages in molecular weight distribution, and the sum of the molecular weight less than 1000Da and the sum of the molecular weight less than 2000 Da are the highest. The content of free amino acid in the collagen enzymolysis product is analyzed, and the collagen is hydrolyzed by collagen hydrolaseRoThe free amino acids produced by APA hydrolysis account for about 6.53%, which is lower than the ratio of free amino acids in pepsin and trypsin enzymolysis products, indicating collagenRoThe APA hydrolysate is mainly present in the form of peptides.

TABLE 1 preparation of collagen peptide by enzymolysis method molecular weight distribution

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

<110> institute for agricultural product processing of Chinese academy of agricultural sciences

<120> collagen hydrolase, and coding gene, preparation method and application thereof

<130> 2021

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 397

<212> PRT

<213> Rhizopus oryzae CGMCC3.17463

<400> 1

Met Lys Phe Thr Leu Val Ser Ser Cys Val Ala Leu Val Val Met Ala

1 5 10 15

Leu Ser Val Glu Ala Ala Pro Asn Gly Lys Lys Leu Ser Ile Ala Leu

20 25 30

Lys Gln Asn Thr Glu Tyr Lys Pro Ser Ala Pro Ala Ala Val Ala Lys

35 40 45

Ala Ile Ala Lys Tyr Gln Lys His Ala Ile Asn Pro Leu Lys Asn Thr

50 55 60

Pro Ser Gly Ser Ser Ser Thr Glu Gly Thr Gly Val Val Pro Val Thr

65 70 75 80

Asp Tyr Gly Asn Asp Ile Glu Tyr Tyr Gly Asp Val Gln Ile Gly Thr

85 90 95

Pro Pro Gln Asn Phe Lys Ile Asn Phe Asp Thr Gly Ser Ser Asp Leu

100 105 110

Trp Val Ala Ser Thr Leu Cys Ala Ser Cys Thr Ser His Thr Arg Tyr

115 120 125

Asn Pro Asn Lys Ser Ser Thr Tyr Val Lys Asp Gly Arg Pro Trp Ser

130 135 140

Ile Ser Tyr Gly Asp Gly Ser Thr Ala Ser Gly Ile Leu Ala Tyr Asp

145 150 155 160

Thr Val Thr Leu Gly Gly Leu Ala Ile Lys Lys Gln Thr Ile Glu Leu

165 170 175

Ala Gln Lys Glu Ser Ser Ser Phe Ala Ser Asp Pro Ile Asp Gly Leu

180 185 190

Leu Gly Leu Gly Phe Asn Thr Ile Thr Thr Val Arg Gly Ile Lys Thr

195 200 205

Pro Val Asp Asn Leu Ile Ser Gln Gly Leu Ile Thr Ser Pro Ile Tyr

210 215 220

Gly Val Ser Leu Gly Lys Ala Ser Asn Gly Gly Gly Gly Glu Tyr Leu

225 230 235 240

Phe Gly Gly Tyr Asn Lys Ser Lys Phe Thr Gly Thr Leu Lys Thr Val

245 250 255

Pro Val Asp Asn Ser Gln Gly Phe Trp Gly Ile Thr Val Ser Asp Leu

260 265 270

Lys Val Gly Thr Lys Ser Tyr Gly Thr Phe Asp Gly Ile Leu Asp Thr

275 280 285

Gly Thr Thr Leu Leu Leu Phe Pro Thr Ala Tyr Ala Asn Lys Val Ala

290 295 300

Thr Ala Tyr Gly Ala Thr Ala Asn Gly Asp Gly Thr Tyr Asn Ile Asn

305 310 315 320

Cys Asn Thr Ser Gly Phe Lys Pro Leu Glu Phe Thr Ile Asn Gly Ala

325 330 335

Thr Phe Tyr Val Pro Thr Asn Ser Leu Ile Phe Gln Lys Ser Gly Ser

340 345 350

Arg Cys Tyr Ala Ser Phe Gly Ser Ser Asn Ile Pro Phe Ala Ile Leu

355 360 365

Gly Asp Thr Phe Leu Lys Asn Asn Tyr Val Val Phe Asn Gln Gln Val

370 375 380

Pro Glu Val Gln Ile Ala Ala Ser Val Asp Gln Asn Lys

385 390 395

<210> 2

<211> 1252

<212> DNA

<213> Rhizopus oryzae CGMCC3.17463

<400> 2

atgaaattca ctcttgtctc ttcttgtgtg gcactggttg tcatggctct ttctgttgaa 60

gcagctccta atggcaagaa actttccatt gctttaaagc aaaatactga atacaagcct 120

agtgctcccg ctgctgttgc aaaggccatt gccaagtatc aaaagcatgc tattaatcct 180

ctcaaaaaca ctccttctgg atcttcctct actgaaggta ctggtgttgt acctgtcact 240

gattacggaa atgatattga atattacggt gatgttcaaa tcggtactcc tcctcaaaac 300

ttcaagatta actttgatac cggttcctcc gatttatggg ttggtaagta caattctctt 360

tttttgttta aaatattata taactaaact attattatta gcctctactt tgtgtgcttc 420

ttgtaccagt catactcgtt acaatcccaa caaatcaagc acttatgtca aggatggtcg 480

tccatggtct atctcttacg gtgatggatc tactgctagc ggtattttag cttacgatac 540

tgttacttta ggtggccttg ctatcaagaa acaaactatt gaattagctc aaaaagaatc 600

cagcagtttc gcttctgatc ctattgatgg tcttctcggt cttggtttca ataccattac 660

cactgttaga ggtatcaaga ctcctgttga taacttgatc agtcaaggtt taattacttc 720

tcctatttat ggtgtttctc tcggtaaggc cagcaatggt ggaggtggtg aatacctctt 780

tggtggttac aataagtcca agttcactgg tactttaaag actgttcctg ttgataactc 840

tcaaggtttc tggggtatta ctgtcagtga tcttaaggtt ggtaccaaga gctatggtac 900

tttcgatggc atccttgata ccggtaccac tcttttactt ttccctactg cctatgccaa 960

caaggtcgcc actgcttatg gtgctactgc taatggtgat ggtacttaca acatcaactg 1020

taacacttct ggtttcaagc ctcttgaatt cactatcaat ggtgctactt tctatgttcc 1080

taccaactct ttgatcttcc aaaagagtgg atccagatgt tatgcttcat tcggttcatc 1140

caacattcct ttcgctattc ttggtgatac tttcttgaag aacaactatg ttgtattcaa 1200

ccaacaagtc cctgaagttc aaatcgctgc ttctgttgac caaaacaaat aa 1252

<210> 3

<211> 58

<212> DNA

<213> Rhizopus oryzae CGMCC3.17463

<400> 3

gtaagtacaa ttctcttttt ttgtttaaaa tattatataa ctaaactatt attattag 58

<210> 4

<211> 1194

<212> DNA

<213> Rhizopus oryzae CGMCC3.17463

<400> 4

atgaaattca ctcttgtctc ttcttgtgtg gcactggttg tcatggctct ttctgttgaa 60

gcagctccta atggcaagaa actttccatt gctttaaagc aaaatactga atacaagcct 120

agtgctcccg ctgctgttgc aaaggccatt gccaagtatc aaaagcatgc tattaatcct 180

ctcaaaaaca ctccttctgg atcttcctct actgaaggta ctggtgttgt acctgtcact 240

gattacggaa atgatattga atattacggt gatgttcaaa tcggtactcc tcctcaaaac 300

ttcaagatta actttgatac cggttcctcc gatttatggg ttgcctctac tttgtgtgct 360

tcttgtacca gtcatactcg ttacaatccc aacaaatcaa gcacttatgt caaggatggt 420

cgtccatggt ctatctctta cggtgatgga tctactgcta gcggtatttt agcttacgat 480

actgttactt taggtggcct tgctatcaag aaacaaacta ttgaattagc tcaaaaagaa 540

tccagcagtt tcgcttctga tcctattgat ggtcttctcg gtcttggttt caataccatt 600

accactgtta gaggtatcaa gactcctgtt gataacttga tcagtcaagg tttaattact 660

tctcctattt atggtgtttc tctcggtaag gccagcaatg gtggaggtgg tgaatacctc 720

tttggtggtt acaataagtc caagttcact ggtactttaa agactgttcc tgttgataac 780

tctcaaggtt tctggggtat tactgtcagt gatcttaagg ttggtaccaa gagctatggt 840

actttcgatg gcatccttga taccggtacc actcttttac ttttccctac tgcctatgcc 900

aacaaggtcg ccactgctta tggtgctact gctaatggtg atggtactta caacatcaac 960

tgtaacactt ctggtttcaa gcctcttgaa ttcactatca atggtgctac tttctatgtt 1020

cctaccaact ctttgatctt ccaaaagagt ggatccagat gttatgcttc attcggttca 1080

tccaacattc ctttcgctat tcttggtgat actttcttga agaacaacta tgttgtattc 1140

aaccaacaag tccctgaagt tcaaatcgct gcttctgttg accaaaacaa ataa 1194

<210> 5

<211> 30

<212> DNA

<213> Artificial Synthesis

<400> 5

cggaattcat gaaattcact cttgtctctt 30

<210> 6

<211> 34

<212> DNA

<213> Artificial Synthesis

<400> 6

ttgcggccgc ttatttgttt tggtcaacag aagc 34