阅读说明：本技术 一种细菌蛋白酶及其制备方法和应用 (Bacterial protease and preparation method and application thereof ) 是由 何海伦 黄嘉丰 颜晓涛 殷婷婷 饶海潋 肖逊 于 2021-07-30 设计创作，主要内容包括：本发明涉及一种细菌蛋白酶及其制备方法和应用,本发明所述的细菌蛋白酶为假交替单胞菌(Pseudoalteromonas sp.)CSN423-M,保藏编号为CCTCC No.M 2015024所产的胞外蛋白酶E423。采用发酵罐发酵工艺制备,包括斜面培养、种子培养和发酵罐发酵。利用本发明发酵培养基制备的发酵液提高了产酶量,缩短了产酶时间,且本发明的蛋白酶在胶原蛋白膨胀中,具有优异的效果。本发明为实现海洋菌蛋白酶的应用及胶原蛋白膨胀改性奠定了基础,具有巨大的经济效益和市场价值。(The invention relates to a bacterial protease and a preparation method and application thereof, wherein the bacterial protease is Pseudoalteromonas (Pseudomonas sp) CSN423-M, and the preservation number is extracellular protease E423 produced by CCTCC No. M024. The preparation method adopts a fermentation tank fermentation process, and comprises slant culture, seed culture and fermentation tank fermentation. The fermentation broth prepared by the fermentation medium improves the enzyme yield, shortens the enzyme production time, and has excellent effect in collagen expansion. The invention lays a foundation for realizing the application of the marine mycoprotease and the expansion modification of the collagen, and has great economic benefit and market value.)

1. A bacterial protease E423, wherein the bacterial protease is an extracellular protease E423 produced by Pseudoalteromonas (Pseudomonas sp.) CSN423-M with the preservation number of CCTCC No. M2015024.

2. The method of claim 1, wherein the bacterial protease E423 is prepared by fermentation of Pseudoalteromonas (Pseudomonas sp.) CSN 423-M; preferably: the fermentation medium comprises the following components in percentage by mass: 1.40-1.45% of corn flour, 3.60-3.65% of bean flour, 1.8-2.1% of casein, CaCl₂ 0.13-0.18％、K₂HPO₄ 0.2-0.4％、KH₂PO₄0.08-0.14 percent of water, and adjusting the pH value to 7.5-8.0 by NaOH;

further preferably: 1.41% of corn flour, 3.62% of bean flour, 2% of casein and CaCl₂ 0.16％、K₂HPO₄ 0.3％、KH₂PO₄0.1 percent, water is complemented, and the pH value is adjusted to 8.0 by NaOH.

3. The method of claim 2, wherein the fermentation seeds are inoculated into the fermentor at 4-10% of the volume of the fermentation medium, and the fermentor parameters are: the temperature of the tank is 15-25 ℃, the pressure of the tank is 0.03-0.05MPa, the stirring speed is 380-: 1.0-1: 1.5, the fermentation time is 72-96 h; preferably: the temperature of the tank is 18-22 ℃, the pressure of the tank is 0.04MPa, the stirring speed is 400rpm, the aeration ratio is 1:1.0, and the fermentation time is 72 hours.

4. The method according to claim 2, wherein the culture medium prepared by replacing distilled water with artificial seawater is used for slant culture and seed culture, and the fermentation tank is used for culture; preferably:

(1) slant culture

Inoculating CSN423-M to 2216E solid slant culture medium by a streaking method, and culturing at 16-22 ℃ for 24-48h to obtain rejuvenated bacteria; 2216E solid slant Medium: 5g of peptone, 1g of yeast powder and Fe₂(PO₄)₃0.01g, adding artificial seawater to 1000ml, pH8.0, 15g of agar powder, and sterilizing with high pressure steam;

(2) seed culture

Inoculating the rejuvenated bacteria in the step (1) into an artificial seawater LB liquid seed culture medium, culturing at 16-22 ℃ and 200rpm for 16-20h to obtain fermented seeds;

artificial seawater LB liquid seed culture medium: peptone 1%, yeast powder 0.5%, NaCl 1%, artificial seawater, NaOH adjusted pH to 8.0.

5. Use of the bacterial protease E423 according to claim 1 or the bacterial protease E423 prepared by the preparation method according to any one of claims 2 to 4 for collagen swelling.

6. The use according to claim 5, wherein said collagen comprises: insoluble bovine tendon type I collagen or porcine skin collagen.

7. The use according to claim 6, characterized in that, when the collagen is expanded, the insoluble bovine tendon type I collagen is treated by adding a buffer and the bacterial protease E423, and the pig skin is directly treated by adding the bacterial protease E423; further, the bacterial protease E423 is obtained by centrifuging the fermented enzyme solution 8000-12,000 Xg for 10-20 min.

8. The use according to claim 7,

insoluble bovine tendon type I collagen swelling:

weighing 0.5-2% (w/v) insoluble bovine tendon type I collagen, adding the insoluble bovine tendon type I collagen into PBS buffer solution with the concentration of 20-50mM and the pH of 7.4, then adding 5-15% of protease E423 enzyme solution prepared by fermentation, wherein the enzyme activity in the total reaction system is 100 and 200U/ml, and incubating for 1-5h at the temperature of 30-40 ℃.

Swelling the pigskin collagen:

cutting pigskin into small pieces of 1-2cm × 1-2cm, soaking in protease E423 enzyme solution prepared by fermentation, with enzyme activity of 100-.

9. The use of claim 5, further comprising reacting the bacterial protease E423 with collagen and a complex polysaccharide to produce a collagen-complex polysaccharide complex sponge.

10. The use according to claim 9, wherein the preparation of the collagen-complex polysaccharide complex sponge comprises the steps of:

(1) preparing a compound polysaccharide stock solution: preparing 1.5-2.5% of composite polysaccharide stock solution by using distilled water;

(2) obtaining collagen stock solution: extracting pig collagen with acid to obtain collagen solution with concentration of 20-40 g/L;

(3) preparing a collagen-composite polysaccharide composite sponge: taking collagen stock solution, composite polysaccharide stock solution and 10-15% glutaraldehyde solution as 3-5: 1, then adding 5-15% protease E423 enzyme solution prepared by fermentation, pouring the mixture into a mold after uniformly mixing the enzyme activity range of 100 plus materials and 200U/ml in the total reaction system, defoaming, fixing the mixture into a mold, precooling the mold, and freeze-drying the mold to obtain the modified collagen-polysaccharide composite sponge.

Technical Field

The invention relates to a bacterial protease, a fermentation tank fermentation process thereof and application of the bacterial protease in collagen expansion, and belongs to the technical field of microbial fermentation and enzyme application.

Background

Collagen is one of the most abundant protein types in animals, and is the most important constituent of skins, scales, bones, tendons, cartilages and the like of animals and fishes. The byproducts of animal processing, such as skin, scale, and bone, are rich in collagen. The marine organisms are also rich in collagen, and the collagen content of the fish skin of some marine fishes can reach more than 80 percent. The collagen has the characteristics of stable structure, high compactness, good water absorption and moisture retention performance, low immunogenicity, strong biocompatibility, capability of being absorbed and degraded by human bodies and the like, and has important research and application values in the fields of food processing, cosmetic manufacturing, biomedicine and the like.

The application of collagen in the field of food processing.

The collagen is an important functional nutritional food, and can be used as a food additive to enhance the tenderness of meat and improve the protein content of the product. Collagen is also used in the preparation of food film materials, such as collagen casings, inner packaging films, and the like. The hydrolysate gelatin of the collagen is also an important food processing raw material, has low melting point, is easy to dissolve in hot water, forms a stable form with high elasticity after being cooled, and is widely applied to the preparation of candies such as gummy candies, soft candies, nougats candies and the like as a candy additive. The application of the gelatin in frozen food is also very wide, and the gelatin can be used as a jelly agent for meat quality protection of the frozen food.

Application of collagen in cosmetic field

Due to the excellent physical elasticity and human tissue compatibility of collagen, collagen is widely used for preparing collagen injection to expand and fill human soft tissues. The collagen and collagen hydrolysate thereof contain a large amount of hydrophilic groups such as hydroxyl groups and the like, thus showing extremely strong water and moisture retention performance, and being applicable to the preparation of water retention masks and various essences. A large number of researches find that the collagen protein hydrolysis peptide has extremely strong biological activity, such as antioxidant activity, antibacterial activity, anti-aging activity, skin cell growth promotion and the like.

Application of collagen in biological medicine field

Collagen is the main supporting component of mammalian extracellular matrix, and cells attach and anchor on the surface of the collagen matrix for growth. The research has proved that the matrix collagen can promote the migration and proliferation of fibroblast, effectively induce the proliferation and differentiation of epithelial cell and osteoblast, and promote the healing of skin and the regeneration of osteoblast. Based on the collagen, the collagen is widely applied to the preparation of medical materials such as collagen fillers, surgical collagen dressings, collagen hemostatic sponges, absorbable surgical sutures and the like. In addition, based on the stable structure and biocompatibility of collagen fiber, collagen can be used for preparing scaffold materials for bones, viscera organs and tubular tissues such as trachea, etc.

Although collagen has good application prospects in the preparation of cosmetics and medical materials such as collagen masks, collagen sponges and biological scaffolds, the development and application of cosmetics and medical collagen materials are severely restricted due to the problems that the collagen has compact structure, low porosity, small diameter of a single pore, unsatisfactory pore communication, inconsistent cellular growth in microporous structure and number and the like. Therefore, in order to obtain a collagen material with a more ideal pore structure, a new method must be developed or collagen must be modified and processed to meet the requirements of tissue repair and cell growth on the collagen material.

Disclosure of Invention

The invention screens a Pseudoalteromonas sp.CSN423 from the intertidal zone of Bohai Bay, obtains a mutant strain Pseudoalteromonas sp.CSN423-M of high-yield exoprotease through ultraviolet mutagenesis, and the Pseudoalteromonas sp.CSN423-M is preserved in China Center for Type Culture Collection (CCTCC) in 2015, 1 month and 9 days with the preservation number of CCTCC No.M 2015024. After the pseudoalteromonas mutant strain CSN423-M is cultured for 96 hours, the protease activity can reach 1461.33U/mL, is improved by 342 percent compared with the mutant starting strain pseudoalteromonas CSN423, and can be used for the industrial production of marine protease.

The collagen is widely applied to preparing medical materials such as collagen fillers, surgical collagen dressings, collagen hemostatic sponges, absorbable surgical sutures and the like. Although collagen has good application prospects in the preparation of cosmetics and medical materials such as collagen masks, collagen sponges and biological scaffolds, the development and application of the existing collagen cosmetics and medical collagen materials are influenced due to the problems of compact structure, low porosity, small pore diameter, unsmooth pore communication, incapability of conforming to cell growth and the like of the collagen.

The invention carries out collagen expansion experiment aiming at protease fermentation liquor produced by CSN423-M, and the fermentation liquor is Pseudoalteromonas sp.CSN423-M fermentation liquor. Research shows that Pseudoalteromonas sp.CSN423-M has excellent application prospect in the expansion and adsorption modification of insoluble bovine tendon type I collagen and collagen represented by pigskin.

The invention aims at providing a bacterial protease E423 which is an extracellular protease produced by Pseudoalteromonas (Pseudomonas sp.) CSN423-M (with the preservation number of CCTCC No. M2015024). The protease E423 is separated and purified through ammonium sulfate precipitation, ion exchange and molecular sieve chromatography, then the amino acid sequence of the nitrogen end is determined through sequencing of the nitrogen end, then a primer is designed according to the amino acid sequence, the gene sequence of the protease is obtained through PCR, and the gene sequence is further translated into the amino acid sequence, and the amino acid sequence of the protease is as follows:

MNLSKITIATLAAFTLVQATSAVAANKKYLNQQANINNSAQNGVSSVLMVSPDQLVGLEAGNELVVLKEIKSNNGDTTRRYQQVYNGLPVIGDTVSLTFNNNGQLKRAHGAAVYDISSDIETVTPKLNQKLAVAKGLQKSSAAIKSVGLEKHNEKSQLAIWVDEQGEAHLVYEVSYVTYGSNPSRPYQIIDANSGDVLFSFDNLQHADATGPGGNLKTGKYIYGTDFDSLNVSQTGNNCSMNTTNVKTINLNGGTSGSSAYSFTCPENTFKEINGAYSPLNDAHYFGNVIFNMYNDWIGTPPLSFQLQMRVHYSSNYENAFWDGSAMTFGDGQNTFYPLVSLDVSAHEVSHGFTEQNSGLVYSGKSGGLNEAFSDMAGEAAEFYMKGTNDWLVGKDIFKGNGALRYMNNPTQDGRSIDNQSSYSSGMDVHYSSGVFNKAFYNLATTSGWDTEKAFKVMARANQLYWTASTNWDLAGNGVMDAACDLNYDPSAVKAALSAVGVNSNLSSGSSCGTTTPPAEDEALSNGVTRTGISGSAKEQLFFTLDVPAGASNLVFNTNGGSGDADLYVRFGSKPTLSTYDCNSTTSTSTESCSIGSAQAGTYYVMVEAWQAISGVSLTGSYDGSTGGGVSPINRTESNVSVASGGWTRFTQNLDAGYSSLDISMAGGSGDADLYVNFGSASSTSSYECRPYKNGNVETCTIENPQAGTWYIDLQGYSAASGITLSISAN, see SEQ ID NO. 1.

The amino acid sequence of the P domain (P-domain) of protease E423 is as follows:

FFTLDVPAGASNLVFNTNGGSGDADLYVRFGSKPTLSTYDCNSTTSTSTESCSIGSAQAGTYYVMVEAWQAISGVSLTGSYDGSTGGGVSPINRTESNVSVASGGWTRFTQNLDAGYSSLDISMAGGSGDADLYVNFGSASSTSSYECRPYKNGNVETCTIENPQAGTWYIDLQGYSAASGITLSISAN, see SEQ ID NO. 2.

The structural mode diagram and the electrophoresis diagram of the P-terminal domain expressed by the fusion of the protease E423 and GST protein are shown in FIG. 1.

The second purpose of the invention is to provide the preparation method of the bacterial protease E423, the bacterial protease E423 is prepared by adopting Pseudoalteromonas (Pseudomonas sp.) CSN423-M fermentation; preferably: the fermentation medium comprises the following components in percentage by mass: 1.40-1.45% of corn flour, 3.60-3.65% of bean flour, 1.8-2.1% of casein, CaCl₂ 0.13-0.18％、K₂HPO₄ 0.2-0.4％、KH₂PO₄0.08-0.14 percent of water, and adjusting the pH value to 7.5-8.0 by NaOH;

further preferably: 1.41% of corn flour, 3.62% of bean flour, 2% of casein and CaCl₂ 0.16％、K₂HPO₄ 0.3％、KH₂PO₄0.1 percent, water is complemented, and the pH value is adjusted to 8.0 by NaOH.

Further, the preparation method comprises the following steps of inoculating fermentation seeds into a fermentation tank according to 4-10% of the volume of a fermentation medium, wherein the fermentation tank parameters are as follows: the temperature of the tank is 15-25 ℃, the pressure of the tank is 0.03-0.05MPa, the stirring speed is 380-: 1.5, the fermentation time is 72-96 h; preferably: the temperature of the tank is 18-22 ℃, the pressure of the tank is 0.04MPa, the stirring speed is 400rpm, the aeration ratio is 1:1.0, and the fermentation time is 72 hours.

Further, the preparation method comprises the steps of firstly adopting a culture medium prepared by artificial seawater instead of distilled water to carry out slant culture and seed culture, and finally adopting a fermentation tank to culture

(1) Slant culture

Inoculating CSN423-M to the sample by scribing2216E solid slant culture medium, culturing at 16-22 deg.C for 24-48h to obtain rejuvenated thallus; 2216E solid slant Medium: 5g of peptone, 1g of yeast powder and Fe₂(PO₄)₃0.01g, adding artificial seawater to 1000ml, pH8.0, 15g of agar powder, and sterilizing with high pressure steam;

(2) seed culture

Inoculating the rejuvenated bacteria in the step (1) into an artificial seawater LB liquid seed culture medium, culturing at 16-22 ℃ and 200rpm for 16-20h to obtain fermented seeds;

artificial seawater LB liquid seed culture medium: peptone 1%, yeast powder 0.5%, NaCl 1%, artificial seawater, NaOH adjusted pH to 8.0.

The formula of the artificial seawater comprises: 28.15g of NaCl, MgSO₄·7H₂O 6.92g,KCl 0.67g,MgCl·6H₂O 5.51g,CaCl·H₂O1.45 g, plus ddH₂O to 1000 ml.

The third purpose of the invention is to provide the application of the bacterial protease E423 in collagen expansion.

Further, the collagen comprises: insoluble bovine tendon type I collagen or porcine skin collagen.

Further, when the collagen is expanded, adding the insoluble bovine tendon type I collagen into a buffer solution and bacterial protease E423 for treatment, and directly adding the bacterial protease E423 into the pigskin for treatment; further, the bacterial protease E423 is obtained by centrifuging the fermented enzyme solution 8000-12,000 Xg for 10-20 min.

Further, insoluble bovine tendon type I collagen swells:

weighing 0.5-2% (w/v) insoluble bovine tendon type I collagen, adding the insoluble bovine tendon type I collagen into PBS buffer solution with the concentration of 20-50mM and the pH of 7.4, then adding 5-15% of protease E423 enzyme solution prepared by fermentation, wherein the enzyme activity in the total reaction system is 100 and 200U/ml, and incubating for 1-5h at the temperature of 30-40 ℃.

Further, pigskin collagen swells:

cutting pigskin into small pieces of 1-2cm × 1-2cm, soaking in protease E423 enzyme solution prepared by fermentation, with enzyme activity of 100-.

The application of the invention also comprises the step of reacting the bacterial protease E423 with collagen and compound polysaccharide together to prepare the collagen-compound polysaccharide compound sponge.

The preparation method of the collagen-compound polysaccharide compound sponge comprises the following steps:

(1) preparing a compound polysaccharide stock solution: preparing 1.5-2.5% of composite polysaccharide stock solution by using distilled water;

(2) obtaining collagen stock solution: extracting pig collagen with acid to obtain collagen solution with concentration of 20-40 g/L;

(3) preparing a collagen-composite polysaccharide composite sponge: taking collagen stock solution, composite polysaccharide stock solution and 10-15% glutaraldehyde solution as 3-5: 1, then adding 5-15% protease E423 enzyme solution prepared by fermentation, pouring the mixture into a mold after uniformly mixing the enzyme activity range of 100 plus materials and 200U/ml in the total reaction system, defoaming, fixing the mixture into a mold, precooling the mold, and freeze-drying the mold to obtain the modified collagen-polysaccharide composite sponge.

Further, uniformly mixing the collagen stock solution, the compound polysaccharide stock solution, the 10-15% glutaraldehyde solution and the protease E423 enzyme solution prepared by fermentation in the step (3), pouring the mixture into a plastic mould, placing the plastic mould into a refrigerator at 4-8 ℃, standing the mixture overnight for defoaming, fixing the mixture at-10 to-20 ℃ for moulding, transferring the moulded mixture into an ultra-low temperature refrigerator at-80 ℃ for precooling for 2-3h, and then freeze-drying the moulded mixture at-50 to-40 ℃ for overnight to obtain the modified collagen-polysaccharide compound sponge.

Insoluble bovine tendon type I collagen according to the present invention was purchased from Worthington Biochemical Co. U.S. insoluble bovine tendon type I collagen (code: M3809).

The invention has the following advantages and innovativeness:

the invention establishes a fermentation process for producing the protease E423 in the fermentation tank, is fermentation application of marine fungus high-yield protease strains, and has excellent effect. On the basis of fermentation process of a fermentation tank, the invention further establishes a treatment process of enzymatic collagen expansion, treats insoluble bovine tendon type I collagen and collagen-rich pigskin and other raw materials by using fermentation liquor prepared by the fermentation process of marine protease E423 produced by the fermentation tank, modifies the collagen, and develops modified collagen sponge on the basis to obtain a modified collagen product with a loose structure, high porosity, large single pore diameter and ideal pore communication. In conclusion, the invention lays a foundation for realizing the industrial application of the collagen modified protease E423, provides a new technology and a new method for modifying and improving the quality of the commercial collagen products, and has great economic benefit and market value.

Drawings

FIG. 1 is a schematic diagram and an electrophoretogram of the P-terminal domain expressed by fusion of protease E423 with GST protein;

wherein: structural pattern and electropherogram of P domain expressed by fusion of protease E423 (lanes 1, 2) with GST protein (lane 3).

FIG. 2 is a graph of the swelling of insoluble bovine tendon type I collagen by fermentation broth of the present invention, along with other control treatments;

wherein: the upper diagram: e423 effects on different times, swelling of collagen; the following figures: collagen swelling comparison of different treatments, 1. PBS; 2. (ii) trypsin; 3. urea; 4, E423; p-domain.

FIG. 3 shows that the common collagenase completely degrades insoluble bovine tendon type I collagen;

which comprises the following steps: the commercially available collagenase, CollagenaseI, other bacterial fermentation-produced collagenase JN2, QS 2-3.

FIG. 4 is a scanning electron microscope observation of the insoluble bovine tendon type I collagen through fermentation liquid expansion.

FIG. 5 is a graph showing the effect of fermentation broth of the present invention and control treatment on pigskin swelling;

wherein EmpA is extracellular protease produced by Vibrio anguillarum.

FIG. 6 is a process for preparing a modified collagen sponge from the fermentation broth of the present invention;

wherein the collagen-compound polysaccharide has different concentration ratios of 1: 1-5: 1.

FIG. 7 is an electron microscope observation of the spatial structure of the modified collagen sponge prepared by the fermentation liquid of the present invention and the control treatment.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 fermentation tank fermentation Process of collagen-modified protease-degrading bacteria

The strain Pseudomonas sp.CSN423-M utilizes the fermentation medium to perform fermentation tank fermentation, and comprises the following specific steps:

(1) slant culture

Inoculating Pseudomonas sp.CSN423-M to 2216E solid slant culture medium by adopting a scribing method, and culturing at 20 ℃ for 24h to obtain rejuvenated thallus;

2216E solid slant Medium (in mass percent): peptone 5g, Yeast 1g, Fe₂(PO₄)₃0.01g, 1000mL of artificial seawater, pH8.0, 15g of agar powder, and sterilizing by high-pressure steam;

(2) seed culture

Inoculating the rejuvenated bacteria in the step (1) into an artificial seawater LB liquid seed culture medium, and culturing at 20 ℃ and 200rpm for 20 hours to obtain fermented seeds;

artificial seawater LB liquid seed culture medium: peptone 1%, yeast powder 0.5%, NaCl 1%, artificial seawater 1000mL, NaOH adjusted pH to 8.0.

The formula of the artificial seawater comprises: 28.15g of NaCl, MgSO₄·7H₂O 6.92g,KCl 0.67g,MgCl·6H₂O 5.51g,CaCl·H₂O1.45 g, plus ddH₂O to 1000 ml.

(3) Fermenting in a fermentation tank

Inoculating the fermentation seeds obtained in the step (2) into a 5L fermentation tank according to 6% of the volume of a fermentation medium, wherein the fermentation tank parameters are as follows: the temperature of the tank is 20 ℃, the pressure of the tank is 0.04MPa, the stirring speed is 300-.

The fermentation medium (by mass percent) is as follows:

1.41% of corn flour, 3.62% of bean flour, 2% of casein and CaCl₂ 0.16％、K₂HPO₄ 0.3％、KH₂PO₄0.1 percent, water is added to make up for 1000mL, and the pH value is adjusted to 8.0 by NaOH.

Example 2 use of the collagen-modified protease fermentation broth obtained in the expansion of insoluble bovine tendon type I collagen.

The fermentation liquor of the invention expands collagen by enzyme method and the contrast detection conditions are as follows:

(1) swelling detection of insoluble bovine tendon type I collagen by protease liquid fermented by bacteria CSN423-M and a control:

shoot using a digital camera (Canon). The collagen swelling degree was determined from the swelling area of collagen in the picture using ImageJ 1.46 software (NIH, usa).

Weighing 1% (w/v) insoluble bovine tendon type I collagen, adding into PBS buffer (20mM, pH 7.4), and adding 10% enzyme solution of protease E423 prepared by fermentation (enzyme activity in total system is 150U/ml); the following three control experiments were set up simultaneously:

A. after incubation for 1-5h at 37 ℃, the collagen volume change of different experimental groups was observed, and PBS buffer alone was used as a blank control. It was found that the collagen swelling capacity of protease E423 was stronger with time (FIG. 2).

B. PBS buffer alone was used as a blank control, and 10% of P-terminal domain (0.15. mu.M) of heterologously expressed and purified protease (heterologously expressed using pGEX4T-1 vector) (FIG. 1), 6M urea (known collagen swelling agent, swelling control experimental group), trypsin (enzyme activity 150U/ml) were added to the other control groups,

experiments show that the collagen modified protease E423 has obvious collagen swelling effect, the swelling degree is 3-7 times of that of urea treatment, and PBS buffer solution or trypsin has no swelling effect on collagen (figure 2).

C. Adding commercially available type I collagen protease (enzyme activity 150U/ml in the system), or adding collagenase JN2 or QS2-3 (enzyme activity 150U/ml in the system) produced by other bacteria fermentation,

it was found that common collagenase such as type I collagenase derived from Clostridium, and collagenase JN2 and QS2-3 derived from Vibrio maritima can completely degrade collagen without swelling collagen (FIG. 3).

The structural change of collagen fibers caused by the expansion of the protease E423 and the P-terminal domain thereof was observed by a scanning electron microscope SEM. The SEM results showed that the collagen swollen with the protease E423 was arranged into a loose network structure (fig. 4).

Example 3 use of fermented collagen-modified protease solutions and P-terminal domains in Swine skin expansion

The pigskin is rich in collagen, the fermented collagen modified protease liquid has a good swelling effect on the pigskin, the pigskin is cut into small blocks of 1.5cm multiplied by 1.5cm, the small blocks are respectively soaked in a collagen modified protease E423 solution (the enzyme activity is 200U/ml), a P-domain solution (0.15 mu M) of heterogeneously expressed and purified E423 and protease EmpA from vibrio anguillarum (the enzyme activity is 300U/ml), the pigskin is subjected to warm bath at 25 ℃ for 5h, and the pigskin is washed for 3-5 times by using sterile water. The control group used PBS buffer instead of protease solution, and the other steps were the same.

Compared with a PBS control group, fresh pigskins treated by P-domain and EmpA of proteases E423 and E423 have a more obvious swelling phenomenon and a softer structural texture (figure 5), the treated pigskins are naturally dried for 3-5h, and observation shows that the pigskins treated by the PBS of the control group are obviously dried and hardened and have transparent edges, the pigskins treated by the collagen-modified protease E423, the P-terminal domain and the vibrio anguillarum protease EmpA are obviously superior to the pigskins of the control group in color and texture, wherein the pigskins treated by the protease E423 have the best effect, and the moisture retention performance of the pigskins treated by the protease E423 is obviously improved (figure 5).

Example 4 preparation of modified collagen composite sponge

Distilled water is used for preparing 2 percent of compound polysaccharide stock solution. The collagen stock solution is a collagen solution obtained by extracting pig collagen with acid, and a 30g/L pig collagen solution; taking a porcine collagen stock solution, a compound polysaccharide stock solution and a 15% glutaraldehyde solution which are equal to 1-5: 1, then adding 10% of the protease liquid E423 (with the activity of 150U/ml and after adding the system) prepared by the invention, uniformly mixing the two solutions on a vortex oscillator, pouring the mixture into a plastic mould (with the liquid level being 4mm), placing the mould in a refrigerator at 4 ℃ for standing overnight for defoaming, fixing the mould at 20 ℃ for molding, transferring the mould into an ultra-low temperature refrigerator at-80 ℃ for precooling for 2h, then placing the mould in a freeze dryer for freeze drying at-50 to-40 ℃, and obtaining the modified collagen-polysaccharide compound sponges with different raw material ratios after freeze drying overnight.

The properties of the obtained collagen sponge are different (the ratio of the collagen stock solution to the composite polysaccharide stock solution) by combining the properties of the modified collagen sponge (figure 6) and different concentration ratios of the collagen-composite polysaccharide. The collagen sponge treated by the collagen modified protease E423 and the P-terminal structural domain has a great improvement on the water retention rate. Through observing the spatial structure of the modified collagen sponge by an electron microscope, the modified collagen sponge treated by the protease E423 or the P-terminal domain has more pores and larger spatial contact area (figure 7) compared with the sponge of the PBS control group, and the drug bearing capacity of the collagen sponge can be obviously improved and a large number of attachment points are provided for cell growth after the modified collagen sponge is applied to clinic, so that the wound healing effect is improved.

All protease enzyme activities in the examples of the present invention were determined by the conventional casein hydrolysis method.

Sequence listing

<110> university of south-middle school

<120> bacterial protease, preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 730

<212> PRT

<213> Pseudomonas sp (Pseudomonas sp.)

<400> 1

Met Asn Leu Ser Lys Ile Thr Ile Ala Thr Leu Ala Ala Phe Thr Leu

1 5 10 15

Val Gln Ala Thr Ser Ala Val Ala Ala Asn Lys Lys Tyr Leu Asn Gln

20 25 30

Gln Ala Asn Ile Asn Asn Ser Ala Gln Asn Gly Val Ser Ser Val Leu

35 40 45

Met Val Ser Pro Asp Gln Leu Val Gly Leu Glu Ala Gly Asn Glu Leu

50 55 60

Val Val Leu Lys Glu Ile Lys Ser Asn Asn Gly Asp Thr Thr Arg Arg

65 70 75 80

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

85 90 95

Leu Thr Phe Asn Asn Asn Gly Gln Leu Lys Arg Ala His Gly Ala Ala

100 105 110

Val Tyr Asp Ile Ser Ser Asp Ile Glu Thr Val Thr Pro Lys Leu Asn

115 120 125

Gln Lys Leu Ala Val Ala Lys Gly Leu Gln Lys Ser Ser Ala Ala Ile

130 135 140

Lys Ser Val Gly Leu Glu Lys His Asn Glu Lys Ser Gln Leu Ala Ile

145 150 155 160

Trp Val Asp Glu Gln Gly Glu Ala His Leu Val Tyr Glu Val Ser Tyr

165 170 175

Val Thr Tyr Gly Ser Asn Pro Ser Arg Pro Tyr Gln Ile Ile Asp Ala

180 185 190

Asn Ser Gly Asp Val Leu Phe Ser Phe Asp Asn Leu Gln His Ala Asp

195 200 205

Ala Thr Gly Pro Gly Gly Asn Leu Lys Thr Gly Lys Tyr Ile Tyr Gly

210 215 220

Thr Asp Phe Asp Ser Leu Asn Val Ser Gln Thr Gly Asn Asn Cys Ser

225 230 235 240

Met Asn Thr Thr Asn Val Lys Thr Ile Asn Leu Asn Gly Gly Thr Ser

245 250 255

Gly Ser Ser Ala Tyr Ser Phe Thr Cys Pro Glu Asn Thr Phe Lys Glu

260 265 270

Ile Asn Gly Ala Tyr Ser Pro Leu Asn Asp Ala His Tyr Phe Gly Asn

275 280 285

Val Ile Phe Asn Met Tyr Asn Asp Trp Ile Gly Thr Pro Pro Leu Ser

290 295 300

Phe Gln Leu Gln Met Arg Val His Tyr Ser Ser Asn Tyr Glu Asn Ala

305 310 315 320

Phe Trp Asp Gly Ser Ala Met Thr Phe Gly Asp Gly Gln Asn Thr Phe

325 330 335

Tyr Pro Leu Val Ser Leu Asp Val Ser Ala His Glu Val Ser His Gly

340 345 350

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

355 360 365

Leu Asn Glu Ala Phe Ser Asp Met Ala Gly Glu Ala Ala Glu Phe Tyr

370 375 380

Met Lys Gly Thr Asn Asp Trp Leu Val Gly Lys Asp Ile Phe Lys Gly

385 390 395 400

Asn Gly Ala Leu Arg Tyr Met Asn Asn Pro Thr Gln Asp Gly Arg Ser

405 410 415

Ile Asp Asn Gln Ser Ser Tyr Ser Ser Gly Met Asp Val His Tyr Ser

420 425 430

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

435 440 445

Trp Asp Thr Glu Lys Ala Phe Lys Val Met Ala Arg Ala Asn Gln Leu

450 455 460

Tyr Trp Thr Ala Ser Thr Asn Trp Asp Leu Ala Gly Asn Gly Val Met

465 470 475 480

Asp Ala Ala Cys Asp Leu Asn Tyr Asp Pro Ser Ala Val Lys Ala Ala

485 490 495

Leu Ser Ala Val Gly Val Asn Ser Asn Leu Ser Ser Gly Ser Ser Cys

500 505 510

Gly Thr Thr Thr Pro Pro Ala Glu Asp Glu Ala Leu Ser Asn Gly Val

515 520 525

Thr Arg Thr Gly Ile Ser Gly Ser Ala Lys Glu Gln Leu Phe Phe Thr

530 535 540

Leu Asp Val Pro Ala Gly Ala Ser Asn Leu Val Phe Asn Thr Asn Gly

545 550 555 560

Gly Ser Gly Asp Ala Asp Leu Tyr Val Arg Phe Gly Ser Lys Pro Thr

565 570 575

Leu Ser Thr Tyr Asp Cys Asn Ser Thr Thr Ser Thr Ser Thr Glu Ser

580 585 590

Cys Ser Ile Gly Ser Ala Gln Ala Gly Thr Tyr Tyr Val Met Val Glu

595 600 605

Ala Trp Gln Ala Ile Ser Gly Val Ser Leu Thr Gly Ser Tyr Asp Gly

610 615 620

Ser Thr Gly Gly Gly Val Ser Pro Ile Asn Arg Thr Glu Ser Asn Val

625 630 635 640

Ser Val Ala Ser Gly Gly Trp Thr Arg Phe Thr Gln Asn Leu Asp Ala

645 650 655

Gly Tyr Ser Ser Leu Asp Ile Ser Met Ala Gly Gly Ser Gly Asp Ala

660 665 670

Asp Leu Tyr Val Asn Phe Gly Ser Ala Ser Ser Thr Ser Ser Tyr Glu

675 680 685

Cys Arg Pro Tyr Lys Asn Gly Asn Val Glu Thr Cys Thr Ile Glu Asn

690 695 700

Pro Gln Ala Gly Thr Trp Tyr Ile Asp Leu Gln Gly Tyr Ser Ala Ala

705 710 715 720

Ser Gly Ile Thr Leu Ser Ile Ser Ala Asn

725 730

<210> 2

<211> 189

<212> PRT

<213> Pseudomonas sp (Pseudomonas sp.)

<400> 2

Phe Phe Thr Leu Asp Val Pro Ala Gly Ala Ser Asn Leu Val Phe Asn

1 5 10 15

Thr Asn Gly Gly Ser Gly Asp Ala Asp Leu Tyr Val Arg Phe Gly Ser

20 25 30

Lys Pro Thr Leu Ser Thr Tyr Asp Cys Asn Ser Thr Thr Ser Thr Ser

35 40 45

Thr Glu Ser Cys Ser Ile Gly Ser Ala Gln Ala Gly Thr Tyr Tyr Val

50 55 60

Met Val Glu Ala Trp Gln Ala Ile Ser Gly Val Ser Leu Thr Gly Ser

65 70 75 80

Tyr Asp Gly Ser Thr Gly Gly Gly Val Ser Pro Ile Asn Arg Thr Glu

85 90 95

Ser Asn Val Ser Val Ala Ser Gly Gly Trp Thr Arg Phe Thr Gln Asn

100 105 110

Leu Asp Ala Gly Tyr Ser Ser Leu Asp Ile Ser Met Ala Gly Gly Ser

115 120 125

Gly Asp Ala Asp Leu Tyr Val Asn Phe Gly Ser Ala Ser Ser Thr Ser

130 135 140

Ser Tyr Glu Cys Arg Pro Tyr Lys Asn Gly Asn Val Glu Thr Cys Thr

145 150 155 160

Ile Glu Asn Pro Gln Ala Gly Thr Trp Tyr Ile Asp Leu Gln Gly Tyr

165 170 175

Ser Ala Ala Ser Gly Ile Thr Leu Ser Ile Ser Ala Asn

180 185