Chitosanase with high activity and its preparation and application

Abstract

The present invention belongs to microbial fermentation, and in particular refers to a chitosanase with high activity and its preparation and application. The chitosanase production strain is inoculated into a sterilized fermentation medium containing a carbon source, a nitrogen source and necessary nutrients, the pH of the fermentation medium is adjusted to 35°C-37°C for ventilation fermentation; during the fermentation, ammonia water is added to control the pH, and glucose solution is added to control the glucose concentration in the medium; when the OD of the fermentation medium reaches 1.5, the pH of the fermentation medium is adjusted to 1.5, and the glucose concentration in the medium is adjusted to 1.5. 600 When the temperature is ≥30, an inducer is added to induce the culture and the fermentation temperature is reduced to 30°C-33°C; when the enzyme activity in the fermentation broth no longer increases or the fermentation cycle is ≤30 hours, the fermentation is completed to prepare a fermentation broth containing chitosanase, and a crude enzyme solution containing chitosanase is obtained by high-pressure homogenization and centrifugation. The present invention solves the problem of low chitosanase activity in the prior art, and has the advantages of high chitosanase activity, small molecular weight of degraded chitosan oligosaccharides, simple fermentation and degradation processes, etc.

Description

Technical Field

[0001] The invention belongs to microbial fermentation, and particularly refers to a chitosanase with high activity and its preparation and application. Background Art

[0002] Chitosan oligosaccharide, also known as amino glucosidase, is a functional oligosaccharide with a degree of polymerization of 2-20 obtained by biodegradation of chitosan from shrimp and crab shells through β-1-4 glycosidic bonds. It is the only positively charged, cationic alkaline amino oligosaccharide in nature. Chitosan oligosaccharide has unique physiological activity and functionality, and has the functions of antibacterial, anti-cancer, lowering blood sugar and blood lipids, regulating endocrine, and eliminating heavy metals. Due to its good water solubility, great functional effects, and high biological activity, research on the production and application of chitosan oligosaccharide has received more and more attention in recent years.

[0003] Common methods for preparing chitosan oligosaccharides include physical degradation, chemical method and enzyme method. The physical degradation method has an incomplete reaction and low yield; the chemical degradation method is easy to hydrolyze a large amount of amino glucosamine monomers, and chemical reagents are easy to cause equipment corrosion and environmental pollution; the enzyme degradation method has a good development prospect because of its mild reaction conditions, easy process control, relatively uniform products, and low environmental pollution.

[0004] Chitosanase is an enzyme that specifically hydrolyzes chitosan, and can catalyze the hydrolysis of the β-1-4 glycosidic bonds of chitosan to obtain chitooligosaccharides. Chitosanase was first discovered by Monaghan in 1973. After nearly 30 years of research, it was found that chitosanase is widely distributed in bacteria, fungi, viruses, plants and other biota.

[0005] The existing literature searched by the applicant includes:

[0006] For example, Zhao Yuqiao et al. optimized the culture conditions of the XK-3 chitosanase strain and obtained a chitosanase activity of 3430U / L. In the patent document with publication number CN102816751A, Bacillus cereus was used for deep liquid fermentation to obtain a crude enzyme activity of 227U / ml and a purified chitosanase preparation with an enzyme activity of 25000U / g.

[0007] Although the existing industrial production of chitosanase has achieved many results, there are still problems such as low enzyme activity, poor application performance, uncontrollable product polymerization degree, and high use cost. In order to further meet the needs of industrial production, it is imperative to develop chitosanase with simple production process, high enzyme activity, and simple and fast chitosan oligosaccharide production. Summary of the invention

[0008] The purpose of the present invention is to provide a chitosanase with high activity and its preparation and application. The chitosanase production strain is selected and the fermentation process is optimized so that the unpurified chitosanase crude enzyme liquid prepared by the present invention has high enzyme activity and can degrade chitosan into chitosan oligosaccharides with an average relative molecular weight of less than 1000.

[0009] The overall technical concept of the present invention is:

[0010] The invention discloses a chitosanase with high activity, characterized in that the unpurified crude enzyme has an enzymatic activity of ≥683U / ml, and the unpurified crude enzyme can degrade chitosan to prepare chitosan oligosaccharides with an average relative molecular weight of ≤1000.

[0011] The method for preparing chitosanase with high activity is to inoculate chitosanase production strains into a sterilized fermentation medium containing a carbon source, a nitrogen source and necessary nutrients to prepare a fermentation liquid containing chitosanase; the method comprises the following steps:

[0012] A. The chitosanase production strain is Escherichia coli, whose Latin name is Escherichia coli and whose deposit number is CGMCC No.19765;

[0013] B. adjusting the pH of the fermentation medium after inoculation to 6.8-7.2, and performing aeration fermentation at a temperature of 35° C.-37° C.; during the fermentation process, controlling the pH to 6.8-7.2 by feeding ammonia water, and feeding glucose solution when the mass percentage concentration of glucose in the fermentation medium is less than 0.5%;

[0014] C. When the OD of the fermentation medium 600 ≥30, adding 0.1mmol / L-0.5mmol / L inducer isopropyl-β-D-thiogalactoside to induce culture, and at the same time reducing the fermentation temperature to 30℃-33℃; when the enzyme activity in the fermentation liquid no longer increases or the fermentation cycle is ≤30 hours, the fermentation is terminated to prepare the fermentation liquid containing chitosanase;

[0015] D. The fermentation liquid containing chitosanase prepared in step C is subjected to high pressure homogenization and centrifugation to obtain a crude enzyme liquid containing chitosanase.

[0016] The Escherichia coli in the present invention, whose Latin name is Escherichia coli and whose preservation number is CGMCC No.19765, is obtained as follows: the applicant obtained a high-yield strain of chitosanase by sampling, separation and purification in Hengshui Lake Wetland in Hengshui City, Hebei Province, and then used genetic engineering technology to express the chitosanase gene in Escherichia coli to obtain a high-yield engineered bacterium of chitosanase, which was submitted to the General Microbiological Center of China National Microbiological Culture Collection Committee located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing for preservation on May 6, 2020. The abbreviation of the preservation unit is CGMCC, and the preservation number of the strain is CGMCC No.19765.

[0017] Application of highly active chitosanase in the preparation of chitooligosaccharides.

[0018] The specific technical concept of the present invention also includes:

[0019] The fermentation medium in step A is composed of the following raw materials:

[0020] Glucose 3.0%~4.0%; yeast powder 2.0%~3.0%; peptone 1.0%~1.5%; sodium chloride 0.8%~1.5%; dipotassium hydrogen phosphate 0.5%~1.5%; potassium dihydrogen phosphate 0.15%~0.3%; magnesium sulfate heptahydrate 0.1%~0.5%; silicone defoaming agent 0.05%~0.15%; the balance is sterile water.

[0021] The mass percentage concentration of the glucose solution added in step B is 0.3% to 0.6%.

[0022] The ventilation condition in step B is a ventilation volume of 0.3 vvm to 0.7 vvm, and a mass percentage concentration of ammonia water is 25% to 28%.

[0023] The condition of high pressure homogenization in step D is a pressure of 45 MPa to 65 MPa.

[0024] The method further comprises a step E, wherein the process conditions of the step are as follows:

[0025] E. The crude enzyme solution containing chitosanase prepared in step D is concentrated by microfiltration and ultrafiltration, and the precipitate is collected by alcohol precipitation to obtain wet-solid enzyme.

[0026] The process conditions of step E are: the crude enzyme solution prepared in step D is passed through a 0.1 micron microfiltration membrane to remove impurities, and then concentrated by 4000 Dalton ultrafiltration to 1 / 8 to 1 / 5 of the original solution, precipitated with 90% to 95% volume percent ethanol or isopropanol, and centrifuged at 6000-10000 rpm to collect the precipitate to obtain wet-solid enzyme.

[0027] The method further comprises a step F, wherein the process conditions of the step are as follows:

[0028] F. The wet enzyme prepared in step E is subjected to low-temperature vacuum drying and low-temperature pulverization to prepare a powdered high-purity chitosanase.

[0029] The process conditions of step F are: vacuum drying the wet enzyme prepared in step E at a temperature of 40°C to 50°C, crushing the dried material at a temperature not exceeding 50°C to prepare powdered high-purity chitosanase, and the enzyme activity of the purified powdered high-purity chitosanase can reach 63000U / g.

[0030] The method also includes a strain activation step, which is to inoculate the chitosanase production strain into LB culture medium and activate it for 10 to 16 hours at a temperature of 35-37°C and a rotation speed of 170-200 rpm.

[0031] The method also includes an expanded culture step, which is to inoculate the activated chitosanase production strain into LB culture medium at a mass percentage of 8% to 12%, and activate it for 10 to 16 hours at a temperature of 35-37°C and a rotation speed of 170-200 rpm.

[0032] The application of highly active chitosanase in the preparation of chitosan oligosaccharides comprises the following process steps:

[0033] a. Prepare a chitosan solution with a mass percentage concentration of 4.0% to 15.0%, adjust the chitosan solution pH to 4.5 to 6.0 with acid, and then heat it to 50°C to 60°C;

[0034] b. preparing the prepared purified chitosanase or the crude enzyme solution containing chitosanase into a chitosanase solution with an enzyme activity of 200u / ml to 300u / ml;

[0035] c. Add the chitosanase solution prepared in step b to the chitosan solution prepared in step a at a ratio of 0.1% to 0.5% by mass, and stir to react for 2 to 5 hours;

[0036] d. The reaction solution after the reaction in step c is heated up and inactivated, and then cooled to room temperature;

[0037] e. The reaction solution prepared in step d is adjusted to pH 7.0-8.0 with sodium hydroxide solution, and subjected to plate and frame filtration, ultrafiltration, nanofiltration, and spray drying to obtain chitosan oligosaccharides with an average relative molecular weight of ≤1000.

[0038] The acid in step a is one of acetic acid, lactic acid, hydrochloric acid or a combination thereof.

[0039] The stirring speed in step c is 20-100 rpm.

[0040] The temperature inactivation conditions in step d are 85-95° C. and 20-40 minutes.

[0041] The mass percentage concentration of the sodium hydroxide solution in step e is 5%-10%.

[0042] In order to verify the technical effect of the present invention, the applicant used the following method to experiment on the chitosanase prepared in the present invention and its application effect:

[0043] 1. The chitosanase activity prepared by the present invention was detected according to the method disclosed in "Chitinase Activity Detection Method" (GB / T 34799-2017);

[0044] 2. The average relative molecular weight of the chitosan oligosaccharide prepared by chitosanase degradation prepared by the present invention was detected according to the method disclosed in "Biostimulant Chitosan Oligosaccharide" (T / CAI 002-2018).

[0045] The substantial features and significant technical advances of the present invention are:

[0046] 1. The present invention uses Escherichia coli (with a preservation number of CGMCC No. 19765) as the production strain of chitosanase, and optimizes the fermentation conditions to make the produced chitosanase have high enzyme activity (unpurified crude enzyme activity can reach 683U / ml), which is much higher than the enzyme activity index of existing products or products reported in the literature.

[0047] 2. The fermentation process of the present invention has a short cycle, a simple production process and mild conditions, which can easily realize industrial production while reducing energy consumption and facilitating control.

[0048] 3. The chitosanase prepared by the present invention can be used in combination with corresponding enzymolysis conditions to efficiently degrade chitosan, greatly improving the yield of chitosan oligosaccharides, and obtaining chitosan oligosaccharides with an average relative molecular weight of less than 1000 and uniform size.

[0049] 4. The method for preparing chitosan oligosaccharides by using chitosanase in the present invention is simple to operate and is green and environmentally friendly, which effectively improves the quality of chitosan oligosaccharide products and can realize the large-scale production of chitosan oligosaccharides.

[0050] 5. The present invention can realize the joint production of chitosanase and chitosan oligosaccharide, reduce the loss of enzyme activity and the occupation of production resources due to chitosanase concentration and drying, and greatly reduce the production cost. DETAILED DESCRIPTION

[0051] The present invention is further described below in conjunction with embodiments, but is not intended to be limiting of the present invention. The protection scope of the present invention shall be based on the contents recorded in the claims, and any replacement of equivalent technical means made according to the description shall not depart from the protection scope of the present invention.

[0052] Example 1

[0053] The chitosanase has high activity, and its unpurified crude enzyme activity is ≥683U / ml. The unpurified crude enzyme can degrade chitosan to prepare chitooligosaccharides with an average relative molecular weight of ≤1000.

[0054] The method for preparing chitosanase with high activity is to inoculate chitosanase production strains into a sterilized fermentation medium containing a carbon source, a nitrogen source and necessary nutrients to prepare a fermentation liquid containing chitosanase; the method comprises the following steps:

[0055] A. The chitosanase production strain is Escherichia coli, whose Latin name is Escherichia coli and whose deposit number is CGMCC No.19765;

[0056] B. adjusting the pH of the fermentation medium after inoculation to 6.8, and performing aeration fermentation at a temperature of 35° C.; during the fermentation process, controlling the pH to 6.8 by feeding ammonia water, and feeding glucose solution when the mass percentage concentration of glucose in the fermentation medium is less than 0.5%;

[0057] C. When the OD of the fermentation medium 600 ≥30, adding 0.1mmol / L inducer isopropyl-β-D-thiogalactoside to induce culture, and at the same time reducing the fermentation temperature to 30°C; when the enzyme activity in the fermentation broth no longer increases or the fermentation cycle is ≤30 hours, the fermentation is terminated to prepare a fermentation broth containing chitosanase;

[0058] D. The fermentation liquid containing chitosanase prepared in step C is subjected to high pressure homogenization and centrifugation to obtain a crude enzyme liquid containing chitosanase.

[0059] The fermentation medium in step A is composed of the following raw materials:

[0060] Glucose 3.0%; yeast powder 2.0%; peptone 1.0%; sodium chloride 0.8%; dipotassium hydrogen phosphate 0.5%; potassium dihydrogen phosphate 0.15%; magnesium sulfate heptahydrate 0.1%; silicone defoamer 0.05%; the balance is sterile water.

[0061] The mass percentage concentration of the glucose solution added in step B is 0.3%.

[0062] The ventilation condition in step B is a ventilation volume of 0.3 vvm and a mass percentage concentration of ammonia water of 25%.

[0063] The condition for high pressure homogenization in step D is a pressure of 45 MPa.

[0064] The method further comprises a step E, wherein the process conditions of the step are as follows:

[0065] E. The crude enzyme solution containing chitosanase prepared in step D is concentrated by microfiltration and ultrafiltration, and the precipitate is collected by alcohol precipitation to obtain wet-solid enzyme.

[0066] The process conditions of step E are: the crude enzyme solution prepared in step D is passed through a 0.1 micron microfiltration membrane to remove impurities, and then concentrated by 4000 Dalton ultrafiltration to 1 / 8 of the original solution, precipitated with 90% volume percent ethanol or isopropanol, and centrifuged at 6000 rpm to collect the precipitate to obtain wet-solid enzyme.

[0067] The method further comprises a step F, wherein the process conditions of the step are as follows:

[0068] F. The wet enzyme prepared in step E is subjected to low-temperature vacuum drying and low-temperature pulverization to prepare a powdered high-purity chitosanase.

[0069] The process conditions of step F are: vacuum drying the wet enzyme prepared in step E at a temperature of 40°C, crushing the dried material at a temperature not exceeding 50°C to prepare powdered high-purity chitosanase, and the enzyme activity of the purified powdered high-purity chitosanase can reach 63000U / g.

[0070] The method also includes a strain activation step, which is to inoculate the chitosanase production strain into LB culture medium and activate it for 10 hours at a temperature of 35° C. and a rotation speed of 170 rpm.

[0071] The method also includes an expanded culture step, in which 8% to 12% of the activated chitosanase production strain is inoculated into LB culture medium at a mass percentage, and activated for 10 to 16 hours at a temperature of 35° C. and a rotation speed of 170 rpm.

[0072] The application of highly active chitosanase in the preparation of chitosan oligosaccharides comprises the following process steps:

[0073] a. Prepare a chitosan solution with a mass percentage concentration of 4.0%, adjust the pH of the chitosan solution to 4.5 with acid, and then heat it to 50°C;

[0074] b. preparing the prepared purified chitosanase or the crude enzyme solution containing chitosanase into a chitosanase solution with an enzyme activity of 200u / ml;

[0075] c. Add the chitosanase solution prepared in step b into the chitosan solution prepared in step a at a ratio of 0.1% by mass, and stir to react for 2 hours;

[0076] d. The reaction solution after the reaction in step c is heated up and inactivated, and then cooled to room temperature;

[0077] e. The reaction solution prepared in step d is adjusted to pH=7.0 with sodium hydroxide solution, and subjected to plate and frame filtration, ultrafiltration, nanofiltration, and spray drying to obtain chitosan oligosaccharides with an average relative molecular weight of ≤1000.

[0078] The acid in the step a is acetic acid.

[0079] The stirring speed in step c is 20 rpm.

[0080] The temperature inactivation conditions in step d are 85° C. and 20 minutes.

[0081] The mass percent concentration of the sodium hydroxide solution in step e is 5%.

[0082] Example 2

[0083] The difference between this embodiment and embodiment 1 is that:

[0084] The method for preparing chitosanase with high activity is to inoculate chitosanase production strains into a sterilized fermentation medium containing a carbon source, a nitrogen source and necessary nutrients to prepare a fermentation liquid containing chitosanase; the method comprises the following steps:

[0085] A. The chitosanase production strain is Escherichia coli, whose Latin name is Escherichia coli and whose deposit number is CGMCC No.19765;

[0086] B. adjusting the pH of the fermentation medium after inoculation to 7.2, and performing aeration fermentation at a temperature of 37° C.; during the fermentation process, the pH was controlled to 7.2 by feeding ammonia water, and when the mass percentage concentration of glucose in the fermentation medium was less than 0.5%, glucose solution was fed into the fermentation medium;

[0087] C. When the OD of the fermentation medium 600 ≥30, adding 0.5mmol / L inducer isopropyl-β-D-thiogalactoside to induce culture, and at the same time lowering the fermentation temperature to 33°C; when the enzyme activity in the fermentation broth no longer increases or the fermentation cycle is ≤30 hours, the fermentation is terminated to prepare a fermentation broth containing chitosanase;

[0088] D. The fermentation liquid containing chitosanase prepared in step C is subjected to high pressure homogenization and centrifugation to obtain a crude enzyme liquid containing chitosanase.

[0089] The fermentation medium in step A is composed of the following raw materials:

[0090] Glucose 4.0%; yeast powder 3.0%; peptone 1.5%; sodium chloride 1.5%; dipotassium hydrogen phosphate 1.5%; potassium dihydrogen phosphate 0.3%; magnesium sulfate heptahydrate 0.5%; silicone defoamer 0.15%; the balance is sterile water.

[0091] The mass percentage concentration of the glucose solution added in step B is 0.6%.

[0092] The ventilation condition in step B is a ventilation volume of 0.7 vvm and a mass percentage concentration of ammonia water of 28%.

[0093] The condition for high pressure homogenization in step D is a pressure of 65 MPa.

[0094] The method further comprises a step E, wherein the process conditions of the step are as follows:

[0095] E. The crude enzyme solution containing chitosanase prepared in step D is concentrated by microfiltration and ultrafiltration, and the precipitate is collected by alcohol precipitation to obtain wet-solid enzyme.

[0096] The process conditions of step E are: the crude enzyme solution prepared in step D is passed through a 0.1 micron microfiltration membrane to remove impurities, and then concentrated by 4000 Dalton ultrafiltration to 1 / 5 of the original solution, precipitated with 95% volume percent ethanol or isopropanol, and centrifuged at 10,000 rpm to collect the precipitate to obtain wet-solid enzyme.

[0097] The method further comprises a step F, wherein the process conditions of the step are as follows:

[0098] F. The wet enzyme prepared in step E is subjected to low-temperature vacuum drying and low-temperature pulverization to prepare a powdered high-purity chitosanase.

[0099] The process conditions of step F are: vacuum drying the wet enzyme prepared in step E at a temperature of 50°C, crushing the dried material at a temperature not exceeding 50°C to prepare powdered high-purity chitosanase, and the purified powdered high-purity chitosanase has an enzyme activity of up to 63,000 U / g.

[0100] The method also includes a strain activation step, which is to inoculate the chitosanase production strain into LB culture medium and activate it for 16 hours at a temperature of 37° C. and a rotation speed of 200 rpm.

[0101] The method also includes an expanded culture step, in which the activated chitosanase production strain is inoculated into LB culture medium at a mass percentage of 12%, and activated for 16 hours at a temperature of 37° C. and a rotation speed of 200 rpm.

[0102] The application of highly active chitosanase in the preparation of chitosan oligosaccharides comprises the following process steps:

[0103] a. Prepare a chitosan solution with a mass percentage concentration of 15.0%, adjust the pH of the chitosan solution to 6.0 with acid, and then heat it to 60°C;

[0104] b. preparing the prepared purified chitosanase or the crude enzyme solution containing chitosanase into a chitosanase solution with an enzyme activity of 300u / ml;

[0105] c. Add the chitosanase solution prepared in step b into the chitosan solution prepared in step a at a ratio of 0.5% by mass, and stir to react for 5 hours;

[0106] d. The reaction solution after the reaction in step c is heated up and inactivated, and then cooled to room temperature;

[0107] e. The reaction solution prepared in step d is adjusted to pH=8.0 with sodium hydroxide solution, and subjected to plate and frame filtration, ultrafiltration, nanofiltration, and spray drying to obtain chitosan oligosaccharides with an average relative molecular weight of ≤1000.

[0108] The acid in the step a is lactic acid.

[0109] The stirring speed in the step c is 100 rpm.

[0110] The temperature inactivation conditions in step d are 95° C. and 40 minutes.

[0111] The mass percent concentration of the sodium hydroxide solution in step e is 10%.

[0112] Example 3

[0113] The difference between this embodiment and embodiment 1 is that:

[0114] The method for preparing chitosanase with high activity is to inoculate chitosanase production strains into a sterilized fermentation medium containing a carbon source, a nitrogen source and necessary nutrients to prepare a fermentation liquid containing chitosanase; the method comprises the following steps:

[0115] A. The chitosanase production strain is Escherichia coli, whose Latin name is Escherichia coli and whose deposit number is CGMCC No.19765;

[0116] B. adjusting the pH of the fermentation medium after inoculation to 7.0, and performing aeration fermentation at a temperature of 36° C.; during the fermentation process, the pH was controlled to be 7.0 by feeding ammonia water, and when the mass percentage concentration of glucose in the fermentation medium was less than 0.5%, glucose solution was fed into the fermentation medium;

[0117] C. When the OD of the fermentation medium 600≥30, adding 0.3mmol / L inducer isopropyl-β-D-thiogalactoside to induce culture, and at the same time reducing the fermentation temperature to 32°C; when the enzyme activity in the fermentation broth no longer increases or the fermentation cycle is ≤30 hours, the fermentation is terminated to prepare a fermentation broth containing chitosanase;

[0118] D. The fermentation liquid containing chitosanase prepared in step C is subjected to high pressure homogenization and centrifugation to obtain a crude enzyme liquid containing chitosanase.

[0119] The fermentation medium in step A is composed of the following raw materials:

[0120] Glucose 3.5%; yeast powder 2.5%; peptone 1.25%; sodium chloride 1.15%; dipotassium hydrogen phosphate 1%; potassium dihydrogen phosphate 0.25%; magnesium sulfate heptahydrate 0.3%; silicone defoamer 0.1%; the balance is sterile water.

[0121] The mass percentage concentration of the glucose solution added in step B is 0.45%.

[0122] The ventilation condition in step B is a ventilation volume of 0.35 vvm, and the mass percentage concentration of ammonia water is 26.5%.

[0123] The condition for high pressure homogenization in step D is a pressure of 55 MPa.

[0124] The method further comprises a step E, wherein the process conditions of the step are as follows:

[0125] E. The crude enzyme solution containing chitosanase prepared in step D is concentrated by microfiltration and ultrafiltration, and the precipitate is collected by alcohol precipitation to obtain wet-solid enzyme.

[0126] The process conditions of step E are: the crude enzyme solution prepared in step D is passed through a 0.1 micron microfiltration membrane to remove impurities, and then concentrated by 4000 Dalton ultrafiltration to 1 / 6 of the original solution, precipitated with 93% volume percent ethanol or isopropanol, and centrifuged at 8000 rpm to collect the precipitate to obtain wet-solid enzyme.

[0127] The method further comprises a step F, wherein the process conditions of the step are as follows:

[0128] F. The wet enzyme prepared in step E is subjected to low-temperature vacuum drying and low-temperature pulverization to prepare a powdered high-purity chitosanase.

[0129] The process conditions of step F are: vacuum drying the wet enzyme prepared in step E at a temperature of 45°C, crushing the dried material at a temperature not exceeding 50°C to prepare powdered high-purity chitosanase, and the enzyme activity of the purified powdered high-purity chitosanase can reach 63000U / g.

[0130] The method also includes a strain activation step, which is to inoculate the chitosanase production strain into LB culture medium and activate it for 13 hours at a temperature of 36° C. and a rotation speed of 185 rpm.

[0131] The method also includes an expanded culture step, in which 10% of the activated chitosanase production strain is inoculated into LB culture medium at a mass percentage, and activated for 13 hours at a temperature of 36° C. and a rotation speed of 185 rpm.

[0132] The application of highly active chitosanase in the preparation of chitosan oligosaccharides comprises the following process steps:

[0133] a. Prepare a chitosan solution with a mass percentage concentration of 9.5%, adjust the pH of the chitosan solution to 5.2 with acid, and then heat it to 55°C;

[0134] b. preparing the prepared purified chitosanase or the crude enzyme solution containing chitosanase into a chitosanase solution with an enzyme activity of 250u / ml;

[0135] c. Add the chitosanase solution prepared in step b to the chitosan solution prepared in step a at a ratio of 0.3% by mass, and stir to react for 3.5 hours;

[0136] d. The reaction solution after the reaction in step c is heated up and inactivated, and then cooled to room temperature;

[0137] e. The reaction solution prepared in step d is adjusted to pH 7.5 with sodium hydroxide solution, and subjected to plate and frame filtration, ultrafiltration, nanofiltration, and spray drying to obtain chitosan oligosaccharides with an average relative molecular weight of ≤1000.

[0138] The acid in step a is one of acetic acid, lactic acid, hydrochloric acid or a combination thereof.

[0139] The stirring speed in the step c is 60 rpm.

[0140] The temperature inactivation conditions in step d are 90° C. and 30 minutes.

[0141] The mass percent concentration of the sodium hydroxide solution in step e is 7.5%.

[0142] Example 4

[0143] The difference between this embodiment and embodiment 1 is that:

[0144] The method for preparing chitosanase with high activity is to inoculate chitosanase production strains into a sterilized fermentation medium containing a carbon source, a nitrogen source and necessary nutrients to prepare a fermentation liquid containing chitosanase; the method comprises the following steps:

[0145] A. The chitosanase production strain is Escherichia coli, whose Latin name is Escherichia coli and whose deposit number is CGMCC No.19765;

[0146] B. adjusting the pH of the fermentation medium after inoculation to 6.9, and performing aeration fermentation at a temperature of 35.5° C.; during the fermentation process, controlling the pH to 6.9 by feeding ammonia water, and feeding glucose solution when the mass percentage concentration of glucose in the fermentation medium is less than 0.5%;

[0147] C. When the OD of the fermentation medium 600 ≥30, adding 0.2mmol / L inducer isopropyl-β-D-thiogalactoside to induce culture, and at the same time lowering the fermentation temperature to 31°C; when the enzyme activity in the fermentation broth no longer increases or the fermentation cycle is ≤30 hours, the fermentation is terminated to prepare a fermentation broth containing chitosanase;

[0148] D. The fermentation liquid containing chitosanase prepared in step C is subjected to high pressure homogenization and centrifugation to obtain a crude enzyme liquid containing chitosanase.

[0149] The fermentation medium in step A is composed of the following raw materials:

[0150] Glucose 3.2%; yeast powder 2.2%; peptone 1.1%; sodium chloride 0.9%; dipotassium hydrogen phosphate 0.8%; potassium dihydrogen phosphate 0.18%; magnesium sulfate heptahydrate 0.2%; silicone defoamer 0.08%; the balance is sterile water.

[0151] The mass percentage concentration of the glucose solution added in step B is 0.3% to 0.6%.

[0152] The ventilation condition in step B is a ventilation volume of 0.4 vvm and a mass percentage concentration of ammonia water of 25.5%.

[0153] The condition for high pressure homogenization in step D is a pressure of 48 MPa.

[0154] The method further comprises a step E, wherein the process conditions of the step are as follows:

[0155] E. The crude enzyme solution containing chitosanase prepared in step D is concentrated by microfiltration and ultrafiltration, and the precipitate is collected by alcohol precipitation to obtain wet-solid enzyme.

[0156] The process conditions of step E are: the crude enzyme solution prepared in step D is passed through a 0.1 micron microfiltration membrane to remove impurities, and then concentrated by 4000 Dalton ultrafiltration to 1 / 6 of the original solution, precipitated with 91% volume percent ethanol or isopropanol, and centrifuged at 7000 rpm to collect the precipitate to obtain wet-solid enzyme.

[0157] The method further comprises a step F, wherein the process conditions of the step are as follows:

[0158] F. The wet enzyme prepared in step E is subjected to low-temperature vacuum drying and low-temperature pulverization to prepare a powdered high-purity chitosanase.

[0159] The process conditions of step F are: vacuum drying the wet enzyme prepared in step E at a temperature of 45°C, crushing the dried material at a temperature not exceeding 50°C to prepare powdered high-purity chitosanase, and the enzyme activity of the purified powdered high-purity chitosanase can reach 63000U / g.

[0160] The method also includes a strain activation step, which is to inoculate the chitosanase production strain into LB culture medium and activate it for 11 hours at a temperature of 35.5° C. and a rotation speed of 175 rpm.

[0161] The method also includes an expanded culture step, in which the activated chitosanase production strain is inoculated into LB culture medium at a mass percentage of 9%, and activated for 11 hours at a temperature of 35.5° C. and a rotation speed of 175 rpm.

[0162] The application of highly active chitosanase in the preparation of chitosan oligosaccharides comprises the following process steps:

[0163] a. Prepare a chitosan solution with a mass percentage concentration of 7.0%, adjust the pH of the chitosan solution to 4.8 with acid, and then heat it to 52°C;

[0164] b. preparing the prepared purified chitosanase or the crude enzyme solution containing chitosanase into a chitosanase solution with an enzyme activity of 220u / ml;

[0165] c. Add the chitosanase solution prepared in step b into the chitosan solution prepared in step a at a ratio of 0.2% by mass, and stir to react for 2.5 hours;

[0166] d. The reaction solution after the reaction in step c is heated up and inactivated, and then cooled to room temperature;

[0167] e. The reaction solution prepared in step d is adjusted to pH=7.2 with sodium hydroxide solution, and subjected to plate and frame filtration, ultrafiltration, nanofiltration, and spray drying to obtain chitosan oligosaccharides with an average relative molecular weight of ≤1000.

[0168] The acid in step a is a combination of acetic acid and lactic acid.

[0169] The stirring speed in the step c is 40 rpm.

[0170] The temperature inactivation conditions in step d are 87° C. and 25 minutes.

[0171] The mass percent concentration of the sodium hydroxide solution in step e is 6%.

[0172] Example 5

[0173] The difference between this embodiment and embodiment 1 is that:

[0174] The method for preparing chitosanase with high activity is to inoculate chitosanase production strains into a sterilized fermentation medium containing a carbon source, a nitrogen source and necessary nutrients to prepare a fermentation liquid containing chitosanase; the method comprises the following steps:

[0175] A. The chitosanase production strain is Escherichia coli, whose Latin name is Escherichia coli and whose deposit number is CGMCC No.19765;

[0176] B. adjusting the pH of the fermentation medium after inoculation to 7.1, and performing aeration fermentation at a temperature of 36.5° C.; during the fermentation process, the pH was controlled to 7.1 by feeding ammonia water, and when the mass percentage concentration of glucose in the fermentation medium was less than 0.5%, glucose solution was fed into the fermentation medium;

[0177] C. When the OD of the fermentation medium 600 When the temperature is ≥30, add 0.4mmol / L inducer isopropyl-β-D-thiogalactoside to induce the culture, and at the same time reduce the fermentation temperature to 32°C; when the enzyme activity in the fermentation broth no longer increases or the fermentation cycle is ≤30 hours, the fermentation is terminated to prepare the fermentation broth containing chitosanase;

[0178] D. The fermentation liquid containing chitosanase prepared in step C is subjected to high pressure homogenization and centrifugation to obtain a crude enzyme liquid containing chitosanase.

[0179] The fermentation medium in step A is composed of the following raw materials:

[0180] Glucose 3.8%; yeast powder 2.8%; peptone 1.4%; sodium chloride 1.4%; dipotassium hydrogen phosphate 1.3%; potassium dihydrogen phosphate 2.28%; magnesium sulfate heptahydrate 0.4%; silicone defoamer 0.13%; the balance is sterile water.

[0181] The mass percentage concentration of the glucose solution added in step B is 0.5%.

[0182] The ventilation condition in step B is a ventilation volume of 0.6 vvm and a mass percentage concentration of ammonia water of 27%.

[0183] The condition for high pressure homogenization in step D is a pressure of 60 MPa.

[0184] The method further comprises a step E, wherein the process conditions of the step are as follows:

[0185] E. The crude enzyme solution containing chitosanase prepared in step D is concentrated by microfiltration and ultrafiltration, and the precipitate is collected by alcohol precipitation to obtain wet-solid enzyme.

[0186] The process conditions of step E are: the crude enzyme solution prepared in step D is passed through a 0.1 micron microfiltration membrane to remove impurities, and then concentrated by 4000 Dalton ultrafiltration to 1 / 7 of the original solution, precipitated with 94% volume percent ethanol or isopropanol, and centrifuged at 9000 rpm to collect the precipitate to obtain wet-solid enzyme.

[0187] The method further comprises a step F, wherein the process conditions of the step are as follows:

[0188] F. The wet enzyme prepared in step E is subjected to low-temperature vacuum drying and low-temperature pulverization to prepare a powdered high-purity chitosanase.

[0189] The process conditions of step F are: vacuum drying the wet enzyme prepared in step E at a temperature of 48°C, crushing the dried material at a temperature not exceeding 50°C to prepare powdered high-purity chitosanase, and the enzyme activity of the purified powdered high-purity chitosanase can reach 63000U / g.

[0190] The method also includes a strain activation step, which is to inoculate the chitosanase production strain into LB culture medium and activate it for 14 hours at a temperature of 36° C. and a rotation speed of 190 rpm.

[0191] The method further comprises an expanded culture step, in which the activated chitosanase production strain is inoculated into LB culture medium at a mass percentage of 11%, and activated for 15 hours at a temperature of 36° C. and a rotation speed of 190 rpm.

[0192] The application of highly active chitosanase in the preparation of chitosan oligosaccharides comprises the following process steps:

[0193] a. Prepare a chitosan solution with a mass percentage concentration of 12.0%, adjust the pH of the chitosan solution to 5.8 with acid, and then heat it to 58°C;

[0194] b. preparing the prepared purified chitosanase or the crude enzyme solution containing chitosanase into a chitosanase solution with an enzyme activity of 280u / ml;

[0195] c. Add the chitosanase solution prepared in step b into the chitosan solution prepared in step a at a ratio of 0.4% by mass, and stir to react for 4.5 hours;

[0196] d. The reaction solution after the reaction in step c is heated up and inactivated, and then cooled to room temperature;

[0197] e. The reaction solution prepared in step d is adjusted to pH 7.8 with sodium hydroxide solution, and subjected to plate and frame filtration, ultrafiltration, nanofiltration, and spray drying to obtain chitosan oligosaccharides with an average relative molecular weight of ≤1000.

[0198] The acid in step a is a combination of lactic acid and hydrochloric acid.

[0199] The stirring speed in the step c is 90 rpm.

[0200] The temperature inactivation conditions in step d are 92° C. and 35 minutes.

[0201] The mass percent concentration of the sodium hydroxide solution in step e is 9%.

[0202] The applicant tested the products prepared in Examples 1-5 and their application effects, and the results are as follows:

[0203] 1. Enzyme activity of crude enzyme solution containing chitosanase

[0204] Crude enzyme activity (U / ml) Purified chitosanase activity (U / g) Chitosanase prepared in Example 1 692 65000 Chitosanase prepared in Example 2 688 64500 Chitosanase prepared in Example 3 696 63800 Chitosanase prepared in Example 4 684 64600 Example 5 Chitosanase prepared 690 63700

[0205] The above tests show that the crude enzyme solution containing chitosanase prepared in Examples 1-5 of the present invention has an average enzyme activity of 690u / ml, and the average enzyme activity after purification is 64300u / g, indicating that the chitosanase prepared in the present invention has a high enzyme activity.

[0206] 2. Degradation effect of chitosan using chitosanase prepared in Examples 1-5

[0207]

[0208] The above tests show that the relative molecular weight of the chitosan oligosaccharides prepared in Examples 1-5 of the present invention is 580-940Da, and the chitosan oligosaccharide yield is about 82.5% on average. The chitosan oligosaccharides prepared by the present invention have small relative molecular weight, high activity, high chitosan oligosaccharide yield, and are suitable for industrial production.

Claims

1. Application of highly active chitosanase in the preparation of chitosan oligosaccharides, characterized in that: The method for preparing chitosanase with high activity is to inoculate chitosanase production strains into a sterilized fermentation medium containing a carbon source, a nitrogen source and necessary nutrients to prepare a fermentation liquid containing chitosanase, comprising the following steps: A. The chitosanase production strain is Escherichia coli, whose Latin name is Escherichia coli and whose deposit number is CGMCC No.19765; B. adjusting the pH of the fermentation medium after inoculation to 6.8-7.2, and performing aeration fermentation at a temperature of 35° C.-37° C.; during the fermentation process, controlling the pH to 6.8-7.2 by feeding ammonia water, and feeding glucose solution when the mass percentage concentration of glucose in the fermentation medium is less than 0.5%; C. When the OD of the fermentation medium 600 ≥30, adding 0.1mmol / L-0.5mmol / L inducer isopropyl-β-D-thiogalactoside to induce culture, and at the same time reducing the fermentation temperature to 30℃-33℃; when the enzyme activity in the fermentation liquid no longer increases or the fermentation cycle is ≤30 hours, the fermentation is terminated to prepare the fermentation liquid containing chitosanase; D. high-pressure homogenization and centrifugation of the fermentation broth containing chitosanase prepared in step C to obtain a crude enzyme solution containing chitosanase; The fermentation medium in step A is composed of the following raw materials: Glucose 3.0%~4.0%; yeast powder 2.0%~3.0%; peptone 1.0%~1.5%; sodium chloride 0.8%~1.5%; dipotassium hydrogen phosphate 0.5%~1.5%; potassium dihydrogen phosphate 0.15%~0.3%; magnesium sulfate heptahydrate 0.1%~0.5%; silicone defoamer 0.05%~0.15%; the balance is sterile water; The mass percentage concentration of the glucose solution added in step B is 0.3% to 0.6%, the ventilation condition in step B is a ventilation volume of 0.3vvm to 0.7vvm, and the mass percentage concentration of the ammonia water is 25% to 28%; The application comprises the following process steps: a. Prepare a chitosan solution with a mass percentage concentration of 4.0% to 15.0%, adjust the chitosan solution pH to 4.5 to 6.0 with acid, and then heat it to 50° C. to 60° C.; the acid in step a is acetic acid, lactic acid, hydrochloric acid or a combination thereof; b. preparing the prepared purified chitosanase or the crude enzyme solution containing chitosanase into a chitosanase solution with an enzyme activity of 200u / ml to 300u / ml; c. Add the chitosanase solution prepared in step b to the chitosan solution prepared in step a at a ratio of 0.1% to 0.5% by mass, and stir to react for 2 to 5 hours; the stirring speed in step c is 20 to 100 rpm; d. The reaction solution after the reaction in step c is heated up and inactivated, and then cooled to room temperature; e. The reaction solution prepared in step d is adjusted to pH 7.0-8.0 with sodium hydroxide solution, and subjected to plate and frame filtration, ultrafiltration, nanofiltration, and spray drying to obtain chitosan oligosaccharides with an average relative molecular weight of 580-940.

2. The use according to claim 1, characterized in that The temperature inactivation conditions in step d are 85-95° C. and 20-40 minutes.

3. The use according to claim 1, characterized in that The mass percentage concentration of the sodium hydroxide solution in step e is 5% to 10%.

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