CN112322533B - Strain for producing efficient collagenase and application thereof - Google Patents

Abstract

The present invention relates to a strain producing high-efficiency collagenase and its application. The strain was preserved in the China Center for Type Culture Collection on October 9, 2020, with the deposit number CCTCC M2020574, and the address is Wuhan University, Wuhan, China. The strain has the ability to degrade collagen, and the produced collagenase can be used to degrade collagen to produce collagen oligopeptide. In particular, it has a strong ability to degrade collagen raw materials such as beef bones and fish skins. It can further enzymatically process by-products of meat processing and fishery processing such as beef bones and fish skins. Cod skins are used as raw materials. Collagen oligopeptide has more than 95% of the peptides with molecular weight less than 3000Da, small molecular weight and high uniformity, easy to be absorbed and utilized by the human body, effectively improving the added value of collagen by-products, and having good economic benefits.

Description

A strain producing high-efficiency collagenase and its application

technical field

The invention relates to a high-efficiency collagenase-producing strain and its application, belonging to the technical field of biotechnology.

Background technique

Collagen is the most abundant protein in animals and a major structural protein in the extracellular matrix. It is widely distributed in connective tissues such as tendons, bones, ligaments, skin and blood vessels of mammals and fish skin and scales of aquatic animals. , fish bone and other tissues, mainly in the form of insoluble fibrous protein. These collagens are often treated as scraps in the food processing process, which not only causes waste of biological resources, but also adversely affects the environment. The full and rational use of these wastes in the food processing process can not only promote the development of livestock and aquatic products processing industries, but also reduce environmental pollution.

The use of collagen raw materials to produce collagen oligopeptides is an important way for high-value utilization of collagen scraps produced in food processing. Collagen oligopeptides are the enzymatic hydrolysis products of collagen, which have various biological functions such as antibacterial, antioxidant and angiotensin-converting enzyme (ACE) inhibitory activities, and have good applications in food, cosmetics, biomedicine and other fields. potential. However, collagen has a right-handed helical supramolecular structure formed by intertwining and folding of three left-handed helical α-peptide chains, which is highly stable and difficult to be degraded. At present, the tool enzymes commonly used in the preparation of collagen oligopeptides in industrial production include plant proteases (bromelain, papain), bacterial proteases (neutral protease, alkaline protease), animal proteases (trypsin, chymotrypsin, collagenase). However, these commercial enzyme preparations often have poor specificity to collagen, and the obtained oligopeptides have a wide molecular weight distribution and high molecular weight, and the product yield is low. Therefore, it is particularly important to screen to obtain new and specific high-efficiency collagenases. The mature microbial fermentation process and product extraction and purification process make microbial-derived collagenase particularly attractive.

At present, collagenase has been obtained from microorganisms such as Bacillus subtilis, Bacillus cereus, Actinomyces, Pseudomonas, but most strains have low enzyme activity or require special induction conditions to produce high-efficiency collagenase. strains are still lacking.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides a strain producing high-efficiency collagenase and its application. The strain belongs to a new species of the family Pseudomonas, and the collagenase produced can be used for the degradation of collagen, thereby increasing the added value of collagen scraps in animal production and aquatic product processing.

The technical scheme of the present invention is as follows:

A new strain of pseudoalteromonas family, Flocculibacter collagenilyticus SM1988, was deposited in the China Center for Type Culture Collection on October 9, 2020, with the deposit number CCTCC M2020574, address: Wuhan University, Wuhan, China.

The strain was isolated from the samples of Pinus spinosa in the intertidal zone (N36.22°, E120.41°) along the coast of Qingdao, Shandong.

According to a preferred embodiment of the present invention, the 16S rDNA gene sequence of the new pseudoalteromonas family Flocculibactercollagenilyticus SM1988 is shown in SEQ ID NO.1.

Preferably according to the present invention, the growth temperature range of the new bacterium Flocculibactercollagenilyticus SM1988 is 10-40°C, and the optimum growth temperature is 25-30°C; the NaCl concentration range for growth is 0.5-6% (w /v), the optimum growth NaCl concentration is 2.5-3.0% (w/v); the growth pH range is 6-10, and the optimum growth pH is 7.0-8.0.

The present invention provides the application of the new bacterium Flocculibacter collagenilyticus SM1988 in the family of Pseudoalteromonas in degrading collagen.

The present invention also provides a method for degrading collagen by utilizing the new bacterium Flocculibactercollagenilyticus SM1988 of the Pseudomonas family, comprising the following steps:

Flocculibacter collagenilyticus SM1988, a new strain of Pseudomonas family, was inoculated into TYS liquid medium for fermentation and culture, and cultured at a shaking table speed of 150 to 200 rpm and a culture temperature of 23 to 27 °C for 4 to 7 days to obtain a fermentation broth; The supernatant obtained by centrifugation of the liquid is the collagenase solution; then add the collagenase solution to the collagen, react at 37-55°C for 2-3 hours, and remove the precipitate after centrifugation, and the obtained supernatant is Collagenase hydrolyzate.

Preferably according to the present invention, the components of the TYS liquid medium are as follows: 5 parts of peptone, 1 part of yeast powder, 30 parts of sea salt, 1000 parts of water, pH 7.0-7.4, all parts by weight.

According to a preferred embodiment of the present invention, the fermentation culture conditions are as follows: the rotating speed of the shaker is 180 rpm, the culture temperature is 25°C, and the culture time is 5-6 d.

Preferably according to the present invention, the centrifugal rotation speed is 6000-8000 r/min, and the centrifugal time is 8-12 min.

Preferably according to the present invention, the collagen is bovine bone collagen or cod skin collagen.

Preferably according to the present invention, the added amount of the collagenase solution is an enzyme-to-material ratio (E/S) of 60-1600 U/g.

Beneficial effects:

The new bacterium Flocculibacter collagenilyticus SM1988 provided by the present invention is a new species of the family Pseudoalteromonas, and has the ability to degrade collagen, and the produced collagenase can be used to degrade collagen to produce collagen oligosaccharides. peptides. In particular, it has a strong ability to degrade collagen raw materials such as beef bones and fish skins. It can further enzymatically process by-products of meat processing and fishery processing such as beef bones and fish skins. Cod skins are used as raw materials. Collagen oligopeptide has more than 95% of the peptides with molecular weight less than 3000Da, small molecular weight and high uniformity, easy to be absorbed and utilized by the human body, effectively improving the added value of collagen by-products, and having good economic benefits.

Description of drawings

Figure 1 is an atomic force microscope (AFM) image and a transmission electron microscope (TEM) image of the new bacterium Flocculibacter collagenilyticus SM1988 of the Pseudomonas family;

In the figure: 1, AFM, 2, TEM.

Figure 2 is a phylogenetic tree diagram constructed from the 16S rDNA sequences of the new Pseudoalteromonas bacterium Flocculibacter collagenilyticus SM1988 and other bacteria.

Fig. 3 is a bidirectional chromatogram of polar lipids of Flocculibacter collagenilyticus SM1988, a new bacterium of the family Pseudoalteromonas family.

Figure 4 is a graph showing the effect of enzymatic hydrolysis of bovine bone collagen by Flocculibacter collagenilyticus SM1988, a new bacterium of the family Pseudoalteromonas family;

In the figure: 1. After degradation, 2. Before degradation.

Figure 5 is a graph showing the effect of enzymolysis on cod skin by Flocculibacter collagenilyticus SM1988, a new bacterium of the family Pseudomonas;

In the figure: A, before degradation, B, after degradation.

FIG. 6 is a broken line graph of the hydrolysis rates of bovine bone collagen (A) and cod skin (B) with different ratios of enzyme materials in Example 6. FIG.

FIG. 7 is a molecular weight distribution diagram of bovine bone collagen and cod skin hydrolyzate in Example 6. FIG.

Detailed ways

The technical solutions of the present invention will be further described below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

Flocculibacter collagenilyticus SM1988, a new strain of Pseudoalteromonas family, was deposited in the China Center for Type Culture Collection on October 9, 2020, with the deposit number CCTCC M 2020574, address: Wuhan University, Wuhan, China.

The raw materials for the preparation of the culture medium in the examples are all commonly used raw materials in the art; both bovine bone collagen and cod skin can be purchased in the market.

Screening and isolation of strains of Example 1

(1) Sample collection

Pine algae were collected from the rocky beach in the intertidal zone (N36.22°, E120.41°) along the coast of Qingdao, placed in an ice box and brought back to the laboratory.

(2) Enrichment and domestication

Gently rinse the algal body with sterilized seawater, cut the algal body into 1/10 TYS liquid medium with sterile scissors, and shake it for 10 min at 25°C and 180 rpm in a shaker to obtain enriched bacteria.

(3) Screening and isolation of strains

The bacterial liquid enriched in step (2) was serially diluted according to the multiples of 10 ¹ , 10 ² , 10 ³ , 10 ⁴ , and 10 ⁵ , and then inoculated on the screening medium plate respectively, and cultivated at 25°C for 48h, The strains that formed the degradation circle were picked out and further purified by plate streak method to obtain strains with the ability to degrade collagen efficiently, which were preserved by the glycerol tube method.

The above-mentioned medium components are as follows:

1/10 TYS liquid medium: 0.5 part of peptone, 0.1 part of yeast powder, 3 parts of sea salt, 100 parts of water, pH 7.0-7.4, all parts by weight.

Screening medium: Each liter of distilled water contains 5 g of peptone, 1 g of yeast powder, 1 g of gelatin, 30 g of sea salt, and 15 g of agar powder, pH 7.0-7.4.

Example 2 Identification of strain morphology

The strains screened and isolated in Example 1 were streaked onto a TYS solid medium plate, and then the plate was inverted and cultured for 24 hours at a temperature of 25°C, and the growth of the colonies on the plate was observed and recorded. (AFM) image and transmission electron microscope (TEM) image, as shown in Figure 1.

It can be seen from Figure 1 that on the TYS medium plate, the colony of this strain is white (yellow in the later stage), round, with neat edges, and the colony is wet and sticky (hard and lumpy in the later stage), indicating that it is smooth, moist and opaque; Red, indicating Gram-negative bacteria; cells in atomic force microscope (AFM) and transmission electron microscope (TEM) images are rod-shaped, with a length and width of 0.8-1.5×0.3-0.5μm, single cells, polar flagella, and can secrete A large number of extracellular polymers.

Example 3 Identification of Physiological and Biochemical Bacteria

The physiological and biochemical characteristics of the strains screened and isolated in Example 1 were identified by routine physiological and biochemical experiments and API 20NE and ZYM reagent strips.

The identification analysis results are shown in Table 1.

Table 1 Comparison of physiological and biochemical characteristics of strains isolated in Example 1 and strains of Pseudomonas family with close kinship

In the table: 1 is the bacterial strain isolated by embodiment 1; 2 is bacterial classification Pseudoalteromonas mariniglutinosaKMM 3635 ^T ; 3 is bacterial classification Psychrosphaera aestuarii PSC101 ^T ; 4 is bacterial classification Psychrosphaeraaquimarina SW33 ^T ; 5 is bacterial classification Psychrosphaera haliotis KDW4 ^T ; 6 is bacterial classification Species Psychrosphaerasaromensis ^SA4-48T ; 7 is bacterial species Salsuginimonas clara LSN- ^49T . + means positive; w means weak positive; - means negative; ND means no data available.

As can be seen from Table 1, the growth temperature range of strain SM1988 screened and isolated in Example 1 is 10-40°C, and the optimum growth temperature is 25-30°C; the growth NaCl concentration range is 0.5-6% (w/v), and the most The NaCl concentration suitable for growth is 2.5-3.0%; the pH range for growth is 6-10, and the optimum pH for growth is 7.0-8.0. Strain SM1988 can reduce nitrate to nitrite, and its oxidase and catalase reactions are positive. The strain can hydrolyze casein, gelatin, Tween 20, Tween40, Tween 60, Tween 80, but cannot hydrolyze agar, starch and esculin.

16S rDNA sequence amplification and identification of the strain of Example 4

The DNA from the strain SM1988 screened and isolated in Example 1 was extracted using the Bio Teke bacterial genome extraction kit, and the specific extraction method was referred to the instructions of the kit.

PCR primers use universal primers:

27F(5'-AGAGTTTGATCCTGGCTCAG-3'),

1492R(5'-GGTTACCTTGTTACGACTTC-3'),

PCR reaction conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30s; annealing at 55°C for 30s; extension at 72°C for 90s; 30 cycles of extension at 72°C for 5 min, and storage at 4°C.

The PCR product was sequenced by BGI, and the sequencing result is shown in SEQ ID NO. 1 of the sequence table, with a length of 1538 bp.

The amplified 16S rDNA sequence was analyzed for homology with the 16S rDNA sequences of all standard strains in the EZBioCloud database. The results showed that the sequences with higher homology belonged to the family Pseudomonas. Representative species in similar families, and the strain Alcanivorax borkumensis Sk2 ^T was used as an exogenous reference for phylogenetic analysis with the strain isolated in Example 1. The MEGA-X software was used to construct a phylogenetic tree using the Neighbor-joining method, as shown in Figure 2.

According to the 16S rDNA sequence alignment results and the biological characteristics of the strain, the strain was identified as a new bacterium of the family Pseudoalteromonas and named as Flocculibacter collagenilyticus SM1988.

Analysis of fatty acid, cytoquinone and polar lipid composition of the strain of Example 5

Using the Sherolock automatic bacterial identification system of MIDI (Microbial Identification) company to analyze the fatty acid composition of the new Pseudomonas bacteria Flocculibacter collagenilyticus SM1988, the specific analysis method refers to the manual of the automatic bacterial identification system, and the analysis results are shown in Table 2. Show.

The polar lipid composition of Flocculibacter collagenilyticus SM1988, which was screened and isolated in Example 1, was analyzed by thin-layer two-dimensional chromatography (TLC). The results are shown in FIG. 3 .

The respiratory quinone fraction of Pseudomonas Flocculibacter collagenilyticus SM1988 was analyzed using an octadecylsilane column (ODS 5 mm, 150 × 4.6 mm) using reversed-phase high performance liquid chromatography.

Table 2 Comparison of fatty acid content between Flocculibacter collagenilyticus SM1988 and strains with similar relationship

In the table: 1 is bacterial species Flocculibacter collagenilyticus; 2 is bacterial species Pseudoalteromonas mariniglutinosa KMM 3635 ^T ; 3 is bacterial species Psychrosphaera haliotis KDW4 ^T.

It can be seen from Table 2 that the main fatty acids of Flocculibacter collagenilyticus SM1988 are: C _18:1 ω7c/C _18:1 ω6c, C _17:1 ω8c, C _16:1 ω7c/C _16:1 ω6c , C _16:0 ; the main respiratory quinone is Q-8, and the main polar lipids are: phosphatidylethanolamine (PE), phosphatidylglycerol (PG). The G+C content of the genomic DNA of Flocculibacter collagenilyticusSM1988, a new bacterium of the family Pseudoalteromonas family, was 39.9 mol%.

Example 6

The application of the new bacterium Flocculibacter collagenilyticusSM1988 of the family Pseudomonas family screened and isolated in Example 1 in the degradation of collagen, including the steps as follows:

Pseudoalteromonas new bacteria Flocculibacter collagenilyticus SM1988 was inoculated into TYS liquid medium for culture, the shaker speed was 180rpm, the culture temperature was 25°C, the culture time was 5d, the fermentation broth was centrifuged at 7000r/min for 10min, and the obtained supernatant The solution is collagenase solution; then add collagenase solution to bovine bone collagen according to the enzyme-material ratio of 1600U/g, and react at 55°C for 2 hours; add the homogenized cod skin according to the enzyme-material ratio of 60U/g. Collagenase solution, react at 37°C for 2h. After the two reactions were completed, the precipitate was removed by centrifugation at 7000 r/min for 10 min, and the obtained supernatant was the collagenase hydrolyzate.

The enzymatic hydrolysis effect of this example is shown in Figures 4 and 5. After the reaction, the amount of the remaining substrate in the reaction was weighed to calculate the enzymatic hydrolysis rate of the collagen substrate. The results are shown in Figure 6.

It can be seen from Figures 4-6 that the collagenase produced by Flocculibacter collagenilyticus SM1988, a new bacterium of the Pseudomonas family, can efficiently hydrolyze bovine bone collagen and cod skin, with the highest enzymatic hydrolysis rates reaching 91.27% and 91.97%, respectively.

In this example, the collagen hydrolyzed solution obtained by degrading the collagen of bovine bone and cod skin was freeze-dried, and then prepared into 5.0 mg/mL collagen with a mobile phase solution (acetonitrile: distilled water: trifluoroacetic acid = 45:55:0.1). After the protease hydrolyzed solution was fully dissolved, it was filtered with a 0.22 μm filter, and the molecular weight distribution of collagen peptides in the enzymatic hydrolyzed solution was analyzed by HPLC. The results are shown in Figure 7 and Table 3.

Table 3 Relative abundance of different molecular weight peptides in bovine bone collagen and cod skin enzymatic hydrolysate

As can be seen from Figure 7 and Table 3, the collagenase secreted by the new bacterium Flocculibactercollagenilyticus SM1988 of the family Pseudomonas family provided by the present invention is used to degrade bovine bone, and the collagen peptides of different molecular weights in the obtained collagenase hydrolyzate are relatively uniformly distributed, wherein Collagen peptides with a molecular weight of less than 5000Da account for more than 40% of the total peptide segments; Flocculibacter collagenilyticus SM1988, a new bacterium of the Pseudomonas family provided by the present invention, is used to degrade cod skin, and the collagenase hydrolyzed solution obtained is mainly low molecular weight less than 3000Da. Molecular weight collagen oligopeptides, accounting for more than 95% of the total peptides. Therefore, the collagenase secreted by Flocculibactercollagenilyticus SM1988 can be used to degrade bovine bone collagen to prepare collagen peptides with different molecular weights, and to degrade cod skin to prepare low molecular weight collagen oligopeptides.

SEQUENCE LISTING

<110> Shandong University

<120> A strain producing high-efficiency collagenase and its application

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<170> PatentIn version 3.5

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<213> Flocculibacter collagenilyticus SM1988

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Claims (10)

1. A new bacterium, Flccculibacter collagegentinicus SM1988, of the pseudoalteromonas family, which is preserved in China center for type culture Collection in 10 months and 9 days of 2020, with the preservation number of CCTCC M2020574, address: china, wuhan university.

2. The new bacterium, Flcculus colagenilyticus SM1988, of the family Pseudoalteromonas according to claim 1, wherein the 16S rDNA gene sequence of the new bacterium, Flcculus colagenilyticus SM1988, of the family Pseudoalteromonas is shown in SEQ ID No. 1.

3. The new bacterium, Flcculus colagenilyticus SM1988, of the family Pseudoalteromonas according to claim 1, wherein the new bacterium, Flcculus colagenilyticus SM1988, of the family Pseudoalteromonas has a growth temperature range of 10-40 ℃, a growth NaCl concentration range of 0.5-6% (w/v), and a growth pH range of 6-10.

4. Use of the new bacterium of the family pseudoalteromonas florigenilyticus SM1988 for the degradation of collagen.

5. A method for degrading collagen using the new bacterium of the family Pseudoalteromonas of claim 1, Flccullibacter collageniltyticus SM1988, comprising the steps of:

inoculating a new bacterium Flccculibacter colagenyticus SM1988 of pseudoalteromonas to a TYS liquid culture medium for fermentation culture, and culturing for 4-7 days at a shaker rotation speed of 150-200 rpm and a culture temperature of 23-27 ℃ to obtain a fermentation liquid; centrifuging the fermentation liquor to obtain supernatant, namely collagen enzyme liquid; and then adding the enzyme solution into the collagen, reacting for 2-3 h at 37-55 ℃, centrifuging, and removing the precipitate to obtain a supernatant, namely the collagen enzymolysis solution.

6. The method of claim 5, wherein said TYS broth comprises the following components: 5 parts of peptone, 1 part of yeast powder, 30 parts of sea salt, 1000 parts of water and 7.0-7.4 of pH, wherein the components are in parts by weight.

7. The method of claim 5, wherein the fermentation conditions are: the rotating speed of a shaking table is 180rpm, the culture temperature is 25 ℃, and the culture time is 5-6 d.

8. The method according to claim 5, wherein the centrifugal rotation speed is 6000 to 8000r/min, and the centrifugal time is 8 to 12 min.

9. The method of claim 5, wherein the collagen is bovine bone collagen or cod skin.

10. The method according to claim 5, wherein the collagenase solution is added in an enzyme-to-material ratio (E/S) of 60 to 1600U/g.

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