CN105670981B

CN105670981B - Serratia marcescens SPG-1 and method for producing chitosanase by using same

Info

Publication number: CN105670981B
Application number: CN201610253272.1A
Authority: CN
Inventors: 赵玉巧; 杜云建
Original assignee: Huaihai Institute of Techology
Current assignee: Huaihai Institute of Techology
Priority date: 2016-04-22
Filing date: 2016-04-22
Publication date: 2020-01-07
Anticipated expiration: 2036-04-22
Also published as: CN105670981A

Abstract

The invention discloses serratia marcescens SPG-1(Serratia marcescens) CCTCC NO: m2015784, the serratia marcescens SPG-1 is gram-negative bacteria, short rod-shaped, single arrangement, no spore, and the size of 0.5-1.0 multiplied by 1.2-2.0 μ M. The invention also discloses a method for preparing chitosanase by adopting the Serratia SPG-1. The chitosanase of the strain has high yield, low culture medium cost, easy control of culture conditions, simple extraction process, energy conservation and environmental protection. The method for producing chitosanase has the advantages of simplicity, practicability and low cost, and is easy to popularize and apply in industrial production.

Description

Serratia marcescens SPG-1 and method for producing chitosanase by using same

Technical Field

The invention relates to a microorganism, in particular to Serratia marcescens SPG-1, and also relates to a method for producing chitosanase by the strain.

Background

Chitosanase is capable of hydrolyzing deacetylated chitosan and is therefore considered to be an enzyme with a specific hydrolytic activity towards linear chitosan. The chitosanase mainly exists in actinomycetes, fungi, bacteria, animals, plants and other biological groups, mainly acts on beta-1.4-glucosaminyl bonds, and degrades chitosan in an endogenous mode to obtain oligomers thereof. Chitosanases were initially classified into class 2, but Fukamizo et al proposed that they be classified into class 3, chitosanase I, chitosanase II, chitosanase III. The first type is capable of hydrolyzing Gen-Gen and Glucina-Gen bonds; the second type can only hydrolyze the Gen-Gen bond; the third category hydrolyzes both Gen-Glen and Gen-Cenacle bonds.

The chitosanase molecular weight separated from plants is 10000 ~ 23000 u, while the chitosanase molecular weight separated from microorganisms is about 20000 ~ 40000 u, most of the chitosanase is alkaline protein, the optimum pH value range of the chitosanase is generally 4.0 ~ 8.0.0, the isoelectric point (pI) change range of the enzyme is also larger, and most of the chitosanase is concentrated on 4.0 ~ 10.1.1.

Chitosan is hydrolyzed by chitosanase to obtain oligosaccharide formed by 2 ~ 10 glucosamine connected by beta-1, 4-glycosidic bond, particularly the typical representation of chitosan oligosaccharide, the main application fields of chitosan oligosaccharide are as follows:

(1) medicine and food additive

The chitosan oligosaccharide has great potential in the aspect of medicine, can prevent the wound from being infected by bacteria, has good ventilation effect and is very helpful for the healing of the wound. Can be degraded by lysozyme to generate natural metabolites, has the characteristics of no toxicity, easy absorption and the like, and has great superiority when being used as a drug sustained-release agent. In 1997, the researchers used low-molecular chitosan for adjuvant therapy, found that the total number of lymphocytes and leukocytes remained stable and the number of T cells increased significantly, and this low-molecular chitosan provided a basis for antitumor research. At present, experiments at a cell level prove that the chitosan oligosaccharide has a killing effect on various tumor cells.

(2) Food product

The application of chitosan oligosaccharide in the food field is more concerned, and the chitosan oligosaccharide has multiple physiological regulation functions, is used as an activating factor of intestinal probiotics, promotes the absorption of calcium and mineral substances, adsorbs heavy metals in vivo and the like. Can replace sodium benzoate and other chemical preservatives in the seasoning, and become a natural green preservative product. It can also reduce the absorption of cholesterol and fat, reduce hypertension, and be widely used in functional beverages for losing weight, expelling toxin, caring skin, regulating immunity, etc. It can be used for coating fresh-keeping of fruits and vegetables, and its coating layer possesses good permeability and water-resisting property, and at the same time possesses the functions of resisting corrosion and resisting bacteria.

(3) Agricultural production

As a novel pesticide, the chitosan oligosaccharide has the characteristics of dual functions of fertilizer efficiency and pesticide effect, is nontoxic and harmless, does not pollute the environment and the like, can change the flora in soil and promote the growth of beneficial microorganisms, can also induce and activate the immune system of plants and improve the resistance of the plants to viruses, has good prevention and treatment effects on diseases such as wheat mosaic disease, rice blast, cotton verticillium wilt, tomato late blight and the like, and can be developed into a growth regulator, a biological pesticide, a fertilizer and the like.

(4) Daily chemical industry

The chitosan oligosaccharide has good moisturizing effect, activates body cells, inhibits the breeding of harmful bacteria on the surface of skin, resists skin diseases, absorbs ultraviolet rays and the like, and is derived from biological extracts, so that the side effects brought by common cosmetics can be well avoided, and the chitosan oligosaccharide can be applied to moisturizing, anti-wrinkle, sun-screening and other types of skin care products; the chitosan oligosaccharide also has permeability for keeping the surface of hair to form a film, is wet and easy to comb, and has the effects of removing dandruff, relieving itching, preventing dust and resisting static electricity.

(5) Biological veterinary drug

By utilizing the antibacterial action, animal diseases caused by staphylococcus aureus and other bacteria are prevented and treated; chitosan oligosaccharide can be used for promoting wound healing, and can be used for adjuvant treatment of trauma or fracture; the carboxymethyl chitosan oligosaccharide has good complexation effect on zinc ions, iron ions, calcium ions and the like, and is expected to be prepared into new natural zinc supplement, iron supplement and calcium supplement.

Disclosure of Invention

The invention aims to solve the technical problem of providing a new serratia marcescens SPG-1 strain capable of producing chitosanase aiming at the defects of the prior art.

The invention also provides a method for producing chitosanase by the Serratia marcescens SPG-1 strain, which aims to solve the problem of high enzyme production cost in the prior art.

The technical problem to be solved by the present invention is achieved by the following technical means. The features of the present invention include the viscous SerratiaBacteria (A), (B)Serratia marcescens) SPG-1 strain itself, and a method for producing chitosanase using the strain.

The strain related to the invention is serratia marcescens SPG-1 (C)Serratia marcescens) Hereinafter, the strain is referred to as Serratia marcescens SPG-1 or strain SPG-1. The strain is preserved in China Center for Type Culture Collection (CCTCC) in 2015 at 12 months and 28 days, and the preservation number is M2015784. And (4) storage address: wuhan university in Wuhan, China, zip code 430072, phone 027 once 68754052.

The invention also relates to the serratia marcescensSerratia marcescens) The method for producing chitosanase from SPG-1 is characterized by comprising the following steps:

(1) preparing strains, namely streaking a serratia marcescens SPG-1 strain on a sterilized rich culture medium plate, performing static culture at 28 ℃ for 1 ~ 2 days, selecting a single colony, performing rich culture medium slant culture for 1 day, and storing the single colony in a refrigerator at 4 ℃ for later use, wherein the rich culture medium comprises 3g of beef extract, 10g of peptone, 5g of NaCl, 15-20 g of agar, 1000mL of water and the pH value is 7.0-7.2;

(2) cell culture and crude enzyme liquid collection, wherein slant culture strain is selected and inoculated into a 250mL triangular flask filled with 50mL liquid culture medium, the strain is cultured for 18 ~ 30h on a shaking table at 25 ~ 30 ℃ and 150 ~ 250 r/min to obtain seed liquid, the seed liquid is inoculated into a 250mL triangular flask containing 30 ~ 50mL liquid culture medium according to the inoculation amount of 5 percent, the seed liquid is cultured for 60 ~ 75h on a shaking table at 26 ~ 30 ℃ and 150 ~ 200 r/min, the seed liquid is taken out, the bacterial precipitation is removed by centrifugation at 8000 r/min, the supernatant is the crude enzyme liquid, the crude enzyme liquid is stored in a refrigerator at 4 ℃ for standby, and the liquid culture medium comprises 25 g of soybean meal, 20g of cane sugar and K₂HPO₄ 2 g，KH₂PO₄2 g，NaCl 5 g，MgSO₄·7H₂0.5 g of O, 1000mL of water and pH of 6.0;

(3) extracting and purifying chitosanase: extracting the crude enzyme solution in the step (2) by using ethanol with the volume of 1 time to obtain crude enzyme precipitate with the specific enzyme activity of 24U/mg protein; the specific enzyme activity of the enzyme obtained by separation and purification of Sephadex G75 is 30U/mgprotein, the relative molecular mass of the chitosanase obtained by separation and purification by SDS-PAGE method is 30000, and the isoelectric point of the chitosanase is 5.98 by isoelectric point coagulation method.

In the technical scheme of the method for producing chitosanase, the further preferable technical characteristics are as follows: in the step (2): the strain cultured on the slant is selected and inoculated into a 250mL triangular flask filled with 50mL liquid culture medium, and the strain is cultured on a shaking table for 24 hours at 25 ℃ at 200 r/min to obtain seed liquid.

In the technical scheme of the method for producing chitosanase, the further preferable technical characteristics are as follows: in the step (2): inoculating the seed solution into a 250mL triangular flask containing 40mL liquid culture medium according to the inoculation amount of 5%, performing shake culture at 28 ℃ for 72h at 180r/min, and taking out.

The invention is further elucidated below.

The invention relates to a screening method of Serratia marcescens SPG-1.

The strain serratia marcescens SPG-1 is obtained by screening according to the following method:

removing impurities in the soil sample, and adding a little of the soil sample into a enrichment medium (colloidal chitosan 5, small molecular chitosan 3, beef extract 2, (NH) containing 5mL₄)₂SO₄ 10，K₂HPO₄ 2，KH₂PO₄ 2，NaCl 5，MgSO₄·7H₂O0.25, water 1000mL, pH 6.5) in a test tube, and culturing for 2 d in a shaking table at the temperature of 30 ℃ and the rotation speed of the shaking table of 160 r/min; collecting a little culture solution after enrichment, diluting and coating with primary sieve culture medium (yeast extract 1, colloidal chitosan 10, powdered chitosan (molecular weight 5 ten thousand) 5, (NH)₄)₂SO₄ 5，K₂HPO₄ 2，NaCl 5，MgSO₄·7H₂0.5O, 15-20 agar, 1000mL water, pH 6.5-7.5) plate, and performing inverted culture at 30 ℃ for 3 d. Then, a single colony was inoculated into a liquid seed medium (peptone 5, yeast extract 5, glucose 5, K)₂HPO₄2，KH₂PO₄ 2，NaCl 5，MgSO₄·7H₂0.5O, 1000mL of water and pH of 6.5-7.5), culturing for 24h on a shaking table at 28 ℃ and 180r/min to obtain a seed solution, and inoculating the seed solution to a fermentation medium (powdered chitosan 10) according to the inoculation amount of 5 percentGlucose 1.0, yeast extract 3, (NH)₄)₂SO₄ 5， K₂HPO₄ 2，NaCl 5，MgSO₄·7H₂O0.5, water 1000mL, pH 6), shaking-culturing at 28 ℃ and 180r/min for 60h, taking out, centrifuging at 8000 r/min to remove thalli precipitate, collecting supernatant as crude enzyme liquid, measuring chitosanase activity by using a DNS method, and finally selecting a chitosanase-producing microorganism from 50 candidate strains: serratia marcescens SPG-1.

Secondly, the property of the Serratia marcescens SPG-1 strain.

Has the following properties:

(1) morphological characteristics: gram-negative bacteria, short rods, in a single array, without spores. The size is 0.5 to 1.0X 1.2 to 2.0 μm, and the movement is not allowed or the mobility is not strong.

(2) The culture characteristics are as follows: the bacterial colony is round, opaque, convex, sticky and easy to pick, is red in color, neat in edge, free of wrinkles, aerobic, uniform and turbid in liquid culture, free of precipitate and bubble generation, and the culture medium is changed from clear yellow to red and then to turbid yellow.

(3) Physiological and biochemical experiment results:

sugar fermentation test results: the strain SPG-1 ferments sucrose, sorbose, ribose, fructose, sorbitol, mannitol, trehalose, rhamnose and glucose, but does not produce gas and ferment xylose, arabinose, lactose and galactose.

② carbon source assimilation test results: see table 1.

TABLE 1 results of carbon source assimilation test of SPG-1 Strain

③ nitrogen source assimilation test result: see table 2.

TABLE 2 Nitrogen source assimilation test results for strain SPG-1

Fourthly, other physiological and biochemical test results: the strain SPG-1 liquefied gelatin is urease-negative, catalase-positive, methyl red experiment negative, V-P experiment result positive, indole experiment result positive, and H is produced₂The S experiment result shows that the bacillus subtilis is strong positive, the milk experiment generates acid coagulation, nitrate is reduced, nitrite is not reduced, the bacillus subtilis is not sensitive to penicillin sodium, the red substance has weak inhibition effect on the bacillus subtilis, and the esculin hydrolysis experiment is positive.

(4) Ecological characteristics

The growth results at different temperatures include that the growth range at the temperature is 10-40 ℃, the red pigment can be normally generated at 15 ~ 30 ℃, and the red pigment is not generated at the temperature above 37 ℃.

② the growth result with different pH values, the growth pH value is 4 ~ 10, the optimum growth pH value is 7 ~ 7.5.5.

③ salt tolerance test: when the NaCl concentration is 2% -8%, the growth is carried out, the pigment production is reduced along with the increase of the salt concentration, and the color is the most red when the NaCl concentration is 2%.

(5) 16S rDNA sequencing results: see table 3.

TABLE 316 SrDNA sequencing results

(6) Evolutionary tree

According to the morphological characteristics, culture characteristics, physiological and biochemical characteristics and ecological characteristics of the strain SPG-1, the strain is mixed with Serratia marcescens (in Bergey's Manual of determinative bacteriology)Serratia marcescens) The classification characteristics of the strain are most consistent, and the 16S rDNA sequencing result is combined, so that the strain SPG-1 is identified to be serratia marcescens and is named as: serratia marcescens SPG-1(Serratia marcescens SPG-1). Sequencing is carried out by Beijing Sanbo Shi remote biotechnology limited company in 6 months of 2013, and sequencing primers are as follows: 27F, sequence No.: 06050080, the sequencing result shows that the homology of the strain SPG-1 and the serratia marcescens is as high as 99%. See figure 1 for the evolutionary tree.

And thirdly, an enzyme production method.

1. Serratia marcescens SPG-1(Serratia marcescens The method for producing chitosanase by SPG-1) comprises the following steps:

(1) preparing strains, namely streaking a serratia marcescens SPG-1 strain on a sterilized rich culture medium plate, performing static culture at 28 ℃ for 1 ~ 2 days, selecting a single colony, performing rich culture medium slant culture for 1 day, and storing the single colony in a refrigerator at 4 ℃ for later use, wherein the rich culture medium comprises 3g of beef extract, 10g of peptone, 5g of NaCl, 15-20 g of agar, 100mL of distilled water and the pH value is 7.0-7.2;

(2) cell culture and crude enzyme liquid collection, wherein slant culture strain is selected and inoculated into a 250mL triangular flask filled with 50mL liquid culture medium, the strain is cultured for 18 ~ 30h on a shaking table at 25 ~ 30 ℃ and 150 ~ 250 r/min to obtain seed liquid, the seed liquid is inoculated into a 250mL triangular flask containing 30 ~ 50mL liquid culture medium according to the inoculation amount of 5 percent, the seed liquid is cultured for 60 ~ 75h on a shaking table at 26 ~ 30 ℃ and 150 ~ 200 r/min and then taken out, the seed liquid is centrifuged at 8000 r/min to remove thallus precipitate, the supernatant is the crude enzyme liquid, and the crude enzyme liquid is stored in a refrigerator at 4 ℃ for standby use, and the liquid culture medium comprises 25 g of soybean meal, 20g of cane sugar and K₂HPO₄ 2 g，KH₂PO₄2 g，NaCl 5 g，MgSO₄·7H₂O0.5 g, water 1000mL, pH 6.0.

(3) Extracting and purifying chitosanase: and (3) extracting the crude enzyme solution in the step (2) by using ethanol with the volume being 1 time, so as to obtain crude enzyme precipitate with the specific enzyme activity of 24U/mg protein. The specific enzyme activity of the enzyme obtained by separation and purification of Sephadex G75 is 30U/mgprotein, the relative molecular mass of the chitosanase obtained by separation and purification by SDS-PAGE method is 30000, and the isoelectric point of the chitosanase is 5.98 by isoelectric point coagulation method.

Compared with the prior art, the invention provides a new serratia marcescens SPG-1 strain capable of producing chitosanase. The chitosanase of the strain has high yield, low culture medium cost, easy control of culture conditions, simple extraction process, energy conservation and environmental protection. The method for producing chitosanase has the advantages of simplicity, practicability and low cost, and is easy to popularize and apply in industrial production.

Drawings

FIG. 1 is a strain clade of Serratia marcescens SPG-1 of the present invention.

Detailed Description

The following further describes particular embodiments of the present invention to facilitate further understanding of the present invention by those skilled in the art, and does not constitute a limitation to the right thereof.

Example 1A Serratia marcescens (II)Serratia marcescens) SPG-1, the preservation number of the strain is CCTCC NO: m2015784. The strain has the following characteristics:

(3) Physiological and biochemical characteristics: sodium citrate, sucrose, glucose, maltose, galactose, trehalose, ribose, D-fructose, sorbitol and mannitol can be used as a unique carbon source, and soluble starch, cellulose, arabinose, L-sorbose, lactose and D-xylose are not used as a unique carbon source; urea, casein, potassium nitrate, ammonium chloride, ammonium sulfate, glycine and aspartic acid can be used as the only nitrogen source, and L-valine is not used as the only nitrogen source. The strain SPG-1 ferments sucrose, sorbose, ribose, fructose, sorbitol, mannitol, trehalose, rhamnose and glucose, but does not produce gas and ferment xylose, arabinose, lactose and galactose.

The strain SPG-1 liquefied gelatin is urease-negative, catalase positive, methyl red test negative, V-P test positive, indole test positive, and produces H₂The S experiment result is positive, the milk experiment generates acid coagulation, nitrate is reduced, nitrite is not reduced, the esculin hydrolysis experiment is positive, the bacillus subtilis is insensitive to penicillin sodium, the bacillus subtilis generates a red substance which is almost insoluble in water but soluble in ethanol, and the red substance has weak inhibition effect on the bacillus subtilisThe application is as follows.

Example 2, a Serratia marcescens strain as described in example 1(Serratia marcescens) The method for producing chitosanase from SPG-1 comprises the following steps:

(1) preparation of strains: the serratia marcescens SPG-1 strain is streaked on a sterilized rich culture medium plate, and after the strain is statically cultured for 1 day at the temperature of 28 ℃, a single colony is picked to carry out the slant culture of the rich culture medium for 1 day and then is stored in a refrigerator at the temperature of 4 ℃ for standby; the rich culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl, 15g of agar, 100mL of distilled water and pH 7.0;

(2) cell culture and crude enzyme liquid collection, wherein slant culture strains are selected and inoculated into a 250mL triangular flask filled with 50mL liquid culture medium, the strain is cultured on a shaking table at 25 ℃ and 150r/min for 18 ~ 30h to obtain seed liquid, the seed liquid is inoculated into the 250mL triangular flask containing 30mL liquid culture medium according to the inoculation amount of 5 percent, the seed liquid is taken out after shaking table culture at 26 ℃ and 150r/min for 60h, the bacterial precipitation is centrifugally removed at 8000 r/min, the supernatant is the crude enzyme liquid which is stored in a refrigerator at 4 ℃ for standby, and the liquid culture medium comprises 25 g of soybean meal powder, 20g of cane sugar and K₂HPO₄ 2 g，KH₂PO₄ 2 g，NaCl 5 g，MgSO₄·7H₂0.5 g of O, 1000mL of tap water, pH 6.0.

Example 3, a Serratia marcescens strain as described in example 1(Serratia marcescens) The method for producing chitosanase from SPG-1 comprises the following steps:

(1) preparation of strains: the serratia marcescens SPG-1 strain is streaked on a sterilized rich culture medium plate, after static culture for 2 days at 28 ℃, single colony is picked out to carry out rich culture medium slant culture for 1 day and then is stored in a refrigerator at 4 ℃ for standby; the rich culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar, 100mL of distilled water and pH 7.2;

(2) culturing cells and collecting crude enzyme solution: selecting a strain cultured on a slant, inoculating the strain into a 250mL triangular flask filled with 50mL of liquid culture medium, culturing for 30 hours on a shaking table at 30 ℃ and 250 r/min to obtain a seed solution, inoculating the seed solution into the 250mL triangular flask containing 50mL of liquid culture medium according to the inoculation amount of 5%, carrying out shaking table culture at 30 ℃ and 200 r/min for 75 hours, taking out, centrifuging at 8000 r/min to remove thallus precipitate, collecting a supernatant which is a crude enzyme solution, and storing in a refrigerator at 4 ℃ for later use; the liquid culture medium is as follows: 25 g of soybean meal, 20g of cane sugar and K₂HPO₄ 2 g，KH₂PO₄ 2 g，NaCl 5 g，MgSO₄·7H₂0.5 g of O, 1000mL of tap water, pH 6.0.

Example 4, a Serratia marcescens strain as described in example 1(Serratia marcescens) In the step (2) of the SPG-1 chitosanase producing method: selecting slant culture strain, inoculating into 250mL triangular flask containing 50mL liquid culture medium, culturing at 25 deg.C for 24 hr on shaking table to obtain seed solution; inoculating the seed solution into a 250mL triangular flask containing 40mL liquid culture medium according to the inoculation amount of 5%, performing shake culture at 28 ℃ for 72h at 180r/min, and taking out. The rest is the same as in example 1.

Example 5A Serratia marcescens SPG-1 (A) as described in example 1Serratia marcescens SPG-1) chitosanase producing process, which includes the following steps:

(1) a large flask with a volume of 1L was filled with 150 mL of fermentation medium (25 g of soybean meal, 20g of sucrose, K)₂HPO₄ 2 g，KH₂PO₄ 2 g，NaCl 5 g，MgSO₄·7H₂0.5 g of O, 1000mL of tap water and 6.0 of pH), inoculating the SPG-1 seed culture solution according to the inoculum size of 5 percent after sterilization, carrying out shake cultivation at 28 ℃ and 165 r/min for 69h, taking out, centrifuging at 8000 r/min to remove thalli precipitate, collecting supernatant fluid which is crude enzyme solution, and storing in a refrigerator at 4 ℃ for later use;

(2) taking 100mL of supernatant, adding 100mL of glacial ethanol, uniformly mixing, standing at 4 ℃ for 5-10 min, freezing and centrifuging at 12000 r/min to obtain crude enzyme precipitate, wherein the specific enzyme activity of the crude enzyme precipitate is 24U/mg protein; dissolving the crude enzyme precipitate in 5mL of acetic acid buffer solution with pH4.4, passing through a Sephadex G75 gel column, eluting with pH4.4 acetic acid buffer solution, collecting fractions, combining the fractions with high enzyme activity, and precipitating with glacial ethanol to obtain enzyme with specific enzyme activity of 69U/mg protein.

Example 6, a Serratia marcescens SPG-1 (C) as described in example 1Serratia marcescens SPG-1) chitosanase producing process, which includes the following steps:

(1) A5L fermenter was charged with 3.5L of fermentation medium (soybean meal 25 g, sucrose 20g, K)₂HPO₄ 2 g，KH₂PO₄ 2 g，NaCl 5 g，MgSO₄·7H₂0.5 g of O, 1000mL of tap water and 6.0 of pH), inoculating the sterilized SPG-1 seed culture solution according to the inoculation amount of 5%, fermenting at the temperature of 28 ℃, the stirring speed of 350r/min and the aeration rate of 0.9 vvm, controlling the pH to be 6.0 in the fermentation process, and discharging the fermentation supernatant until 69 hours until the content of chitosanase in the fermentation supernatant is 3500U/L.

(2) 300mL of supernatant is taken and added with 300mL of glacial ethanol, the mixture is kept stand for 5 ~ 10 min at 4 ℃ after being mixed evenly, crude enzyme precipitate is obtained after being frozen and centrifuged at 12000 r/min, the specific enzyme activity of the crude enzyme precipitate is 35U/mg protein, the crude enzyme precipitate is dissolved in 5mL of acetic acid buffer solution with pH4.4 and passes through a Sephadex G75 gel column, the pH4.4 acetic acid buffer solution is used for eluting and collecting fractions, the fractions with high enzyme activity are combined, and the specific enzyme activity of the enzyme obtained after the precipitation by the glacial ethanol is 76U/mg protein.

Example 7. Serratia marcescens SPG-1 (as described in example 1)Serratia marcescens SPG-1) chitosanase producing method for hydrolyzing chitosan, which comprises the following steps:

(3) And (3) adding 50mg of chitosanase obtained in the step (2) into 100mL of colloidal chitosan solution with the concentration of 20g/L and the pH value of 6, reacting for 5 hours at 45 ℃, and carrying out silica gel layer chromatography to obtain enzyme reaction products, wherein the enzyme reaction products mainly comprise chitosan oligosaccharide, a small amount of glucosamine and the balance of oligosaccharide with different molecular weights.

Claims

1. Serratia marcescens (A)Serratia marcescens) A process for producing chitosanase from SPG-1, characterized in that Serratia marcescens (Serratia marcescens) ((S. marcescens))Serratia marcescens) The preservation number of SPG-1 is CCTCC NO: m2015784; the method for producing chitosanase from SPG-1 comprises the following steps:

(2) culturing cells and collecting crude enzyme solution: the slant culture was picked and inoculated into a 250mL Erlenmeyer flask containing 50mL of liquid mediumCulturing the mixture on a shaking table for 24 hours at 25 ℃ and 200 r/min to obtain a seed solution, inoculating the seed solution into a 250mL triangular flask containing 40mL of liquid culture medium according to the inoculation amount of 5%, carrying out shaking table culture at 28 ℃ and 180r/min for 72 hours, taking out the seed solution, centrifuging at 8000 r/min to remove thalli sediment, collecting supernatant, namely crude enzyme solution, and storing the crude enzyme solution in a refrigerator at 4 ℃ for later use; the liquid culture medium is as follows: 25 g of soybean meal, 20g of cane sugar and K₂HPO₄ 2 g，KH₂PO₄ 2 g，NaCl 5 g，MgSO₄·7H₂0.5 g of O, 1000mL of water and pH of 6.0;