CN111944789B - Method for producing chitinase by fermenting chaetomium globosum and application thereof - Google Patents

Abstract

The invention discloses a method for producing chitinase by fermenting chaetomium globosum and application thereof, which adopts chaetomium globosum HY1 to activate strains, wherein an activation culture medium comprises glucose, peptone, potassium dihydrogen phosphate and disodium hydrogen phosphate; performing shake culture or fermentation tank culture, wherein the fermentation culture medium comprises soluble starch, glycerol, tryptone, yeast powder, corn steep liquor, and NH₄NO₃、MnSO₄、MgSO₄、Na₂HPO₄、K₂HPO₄The chitinase produced by fermentation is used for preparing the chitin oligosaccharide. The culture method is simple, the nutritional requirement is not high, the activity of the chitinase produced by liquid fermentation is high, and the chitinase has a high industrial application value.

Description

Method for producing chitinase by fermenting chaetomium globosum and application thereof

Technical Field

The invention relates to a method for producing chitinase by fermenting chaetomium globosum and application of the chitinase in preparation of chitosan oligosaccharide, belonging to the technical field of biology.

Background

Chitin is one of renewable resources with huge storage capacity in nature, is widely distributed in nature, mostly exists in cell walls of fungi and outer skeletons of arthropods such as shrimps and crabs as biological structure contents, and the storage capacity of the chitin in marine organisms is about 10 hundred million tons. Chitosan oligosaccharide is a degradation product of chitin, and is different from chitin in that the molecular weight is reduced and the polymerization degree is reduced, so that the chitosan oligosaccharide has better water solubility, is easy to be absorbed collectively and can be completely degraded in vivo. The chitosan oligosaccharide has different pharmacological actions and biological activities, and has stronger application prospects in the aspects of improving the immunity of organisms, resisting oxidation, bacteria and tumors. The chitin oligosaccharide which can be directly absorbed by human body is prepared by degrading chitin, and the chitin oligosaccharide becomes a research hotspot for high-additional comprehensive utilization of chitin at present.

Chitin is easier to be absorbed and utilized after being degraded into chitosan oligosaccharide, compared with a chemical degradation method, enzymatic degradation can specifically cut off beta-1, 4 glycosidic bonds on a chitin chain, the process is easy to control, the reaction specificity is strong, the oligosaccharide content of the product is high, and the functionality is better. In addition, the enzymatic degradation reaction condition is mild, the product safety is high, and the environmental pollution is not easy to cause.

Chitinase (EC.3.2.1.14) is an enzyme for specifically degrading chitin, widely exists in bacteria and eukaryotes, but the Chitinase for efficiently degrading chitin is not available in the industrial application field, the enzyme activity is not high, the stability is poor and other characteristic reasons, the application of the Chitinase in the preparation of chitosan oligosaccharide is limited, and the application in the industries of food, agriculture, medicine, environmental protection and the like is further limited.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a culture method for producing high-activity chitinase by fermenting chaetomium globosum and application thereof, wherein the chaetomium globosum HY1 is used as an original strain, and the chitinase is produced by a liquid fermentation method and can be used for producing chitooligosaccharide by efficiently hydrolyzing chitin.

The technical scheme provided by the invention is as follows: a method for producing chitinase by fermentation of chaetomium globosum is characterized by comprising the following specific steps:

(1) strain: chaetomium globosumChaetomiumglobosum，The preservation name is: HY1, depository: china microbial strain preservationThe general microbiological center of the management committee, the preservation number is CGMCC No. 10609;

(2) activating strains: transferring the strain stored on the test tube slant into an activation culture medium, and activating at 28 deg.C and 200rpm until the strain is turbid to obtain activated seed solution; activating a culture medium: 8-15 g/L glucose, 3-6 g/L peptone, 1-3 g/L potassium dihydrogen phosphate, 1-2.5 g/L disodium hydrogen phosphate, and pH 6.5-7.0;

(3) fermentation production of chitinase

1) Fermentation medium: 15-20 g/L of soluble starch, 10-15 g/L of glycerol, 5-10 g/L of tryptone, 2-4 g/L of yeast powder, 4-8 g/L of corn steep liquor and 2-5 g/L of NH₄NO₃，0.5～1.5 g/L MnSO₄，2～4 g/L MgSO₄，1～2.5 g/L Na₂HPO₄，1～3 g/L K₂HPO₄4-8 g/L colloidal chitin, and the pH value is 6.5-7.5;

2) shake cultivation or fermentation tank cultivation:

shake cultivation: inoculating the activated seed liquid into the sterilized fermentation medium according to the inoculation amount of 3-6% (v/v), culturing in a rotary shaking table at 200rpm, wherein the culture temperature is 28-30 ℃, after fermentation for 3-5 days, culturing is finished, and performing centrifugal filtration sterilization to obtain the enzyme liquid;

culturing in a fermentation tank: the method comprises the steps of filling 70% of the culture medium into a fermentation medium according to a volume ratio, sterilizing the culture medium for 20 min by steam at 118 ℃, inoculating activated strains according to a ratio of 3-6% (v/v), controlling the pH value in the fermentation process to be maintained at 6.5-7.5, adjusting the culture temperature to be 28-30 ℃, maintaining the dissolved oxygen at more than 5%, fermenting for 3-5 days, and performing centrifugal filtration sterilization to obtain the enzyme liquid.

The chitinase produced by the fermentation method is applied to the preparation of chitosan oligosaccharide.

The invention has the beneficial effects that:

1. novel biological source, high chitinase activity and great application value

The invention relates to chitinase derived from chaetomium globosum HY1, which has high enzyme activity, high yield of chitosan oligosaccharide prepared by catalytically converting a substrate and great application value.

2. The culture method of the production strain is simple

The culture method of chaetomium globosum HY1 is simple, has low nutritional requirement, is easy to operate, is not easy to be infected with infectious microbes in the fermentation process, does not need a complex material supplementing process, and is suitable for industrial large-scale production.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

Chitinase activity assay used: centrifuging the fermentation liquor at 8000 rpm for 5min to obtain fermentation supernatant, adding 1mL of supernatant enzyme solution with appropriate dilution, adding 0.5g of chitin colloid, diluting to 2 mL, reacting in 40 deg.C water bath for 20 min, adding 3mL of DNS reagent, boiling in water bath for 5min, collecting 1mL of sample solution, adding 3mL of distilled water, and centrifuging at 8000 rpm for 2 min. And taking the centrifuged supernatant as a blank to measure the OD value at 540 nm, and calculating the enzyme activity according to a DNS standard curve. Blank control group: and inactivating 1mL of enzyme solution with proper dilution in a boiling water bath for 5min, adding colloidal chitin solution and DNS with the same volume, and treating by the same method as the sample group. Calculating enzyme activity: the mu mol number of glucosamine generated by hydrolysis in unit volume (mL) and unit time (min) is taken as a definition unit of enzyme activity,

preparation of the colloidal chitin used: weighing 10 g of chitin, adding 40mL of acetone for full infiltration, grinding at 4 ℃ while adding 300 mL of concentrated HCl, standing for 5 h, slowly adding the mixture into 1.5L of 50% ethanol, stirring vigorously while adding, and standing for 2 h to precipitate out colloidal chitin. Removing supernatant, collecting colloidal chitin precipitate, and repeatedly washing with distilled water to neutral.

Configuration of DNS reagents used: 91g of sodium potassium tartrate is added into 500 mL of hot water, stirred and dissolved in a water bath at 45 ℃, then 20g of NaOH and 3.15g of 3, 5-dinitrosalicylic acid (DNS) are sequentially added, then 2.5g of crystalline phenol and 2.5g of anhydrous sodium sulfite are added, stirred and dissolved, cooled, and the volume is adjusted to 1000mL by using distilled water, and the mixture is stored in a brown bottle and placed in a dark place for one week for use.

Example 1: fermentation production of chitinase (1) by culturing Chaetomium globosum HY1 on shaking bed

Selecting a loop from the strain preserved on the test tube slant, transferring into an activation culture medium, and activating at 28 deg.C and 200rpm for 25 hr to obtain activated seed solution. Activating a culture medium: 8g/L glucose, 3 g/L peptone, 1 g/L potassium dihydrogen phosphate, 1 g/L disodium hydrogen phosphate, pH 6.5.

With the preferred fermentation medium: 15 g/L soluble starch, 10 g/L glycerol, 5 g/L tryptone, 2 g/L yeast powder, 4 g/L corn steep liquor and 2 g/L NH₄NO₃，0.5 g/L MnSO₄，2 g/L MgSO₄，1 g/L Na₂HPO₄，1 g/L K₂HPO₄4 g/L colloidal chitin, initial pH 6.5. The sterilized preferable fermentation medium is inoculated with an activated seed culture solution according to the inoculation amount of 3% (v/v), cultured in a rotary shaking table at 200rpm, the culture temperature is 28 ℃, the culture is completed after 3 d of fermentation, and the enzyme solution can be used after centrifugal filtration sterilization. After the fermentation, the activity of the chitinase was determined to be 28.51U/mL.

Example 2: fermentation production of chitinase (2) by culturing Chaetomium globosum HY1 on shaking bed

Transferring the strain stored on the test tube slant into an activation culture medium, and activating at 28 deg.C and 200rpm for 30 hr until the strain is turbid to obtain activated seed solution. Activating a culture medium: 15 g/L glucose, 6 g/L peptone, 3 g/L potassium dihydrogen phosphate, 2.5 g/L disodium hydrogen phosphate, pH 7.0.

Preferred fermentation media are used: 20 g/L soluble starch, 15 g/L glycerin, 10 g/L tryptone, 4 g/L yeast powder, 8g/L corn steep liquor and 5 g/L NH₄NO₃，1.5 g/L MnSO₄，4 g/L MgSO₄，2.5 g/L Na₂HPO₄，3 g/L K₂HPO₄8g/L colloidal chitin, initial pH 7.5. The sterilized preferable fermentation medium is inoculated with the activated seed culture solution according to the inoculation amount of 6% (v/v), cultured in a rotary shaking table at 200rpm, the culture temperature is 30 ℃, the culture is completed after 5d of fermentation, and the enzyme solution can be used after centrifugal filtration sterilization.After the fermentation, the activity of the chitinase is determined to be 25.36U/mL.

Example 3: fermentation production of chitinase (1) by culturing Chaetomium globosum HY1 in fermentation tank (10L)

Transferring the strain stored on the test tube slant into an activation culture medium, and activating at 28 deg.C and 200rpm for 30 hr until the strain is turbid to obtain activated seed solution. Activating a culture medium: 10 g/L glucose, 5 g/L peptone, 2 g/L potassium dihydrogen phosphate, 1.5 g/L disodium hydrogen phosphate, pH 7.0.

Preferred fermentation media are used: 18g/L soluble starch, 12 g/L glycerol, 7 g/L tryptone, 3 g/L yeast powder, 5 g/L corn steep liquor and 4 g/L NH₄NO₃，1 g/L MnSO₄，3 g/L MgSO₄，2 g/L Na₂HPO₄，2 g/L K₂HPO₄6 g/L colloidal chitin. Loading into optimized fermentation medium at 70% volume ratio, steam sterilizing at 118 deg.C for 20 min, inoculating activated strain at 5% (v/v), controlling pH at 6.8 during fermentation, adjusting culture temperature to 28 deg.C, maintaining dissolved oxygen at above 5%, fermenting for 3.5d, centrifuging, filtering, and sterilizing to obtain enzyme solution. After the fermentation, the chitinase activity was determined to be 39.73U/mL.

Example 4: fermentation production of chitinase (2) by culturing Chaetomium globosum HY1 in fermentation tank (10L)

Transferring the strain stored on the test tube slant into an activation culture medium, and activating at 28 deg.C and 200rpm for 30 hr until the strain is turbid to obtain activated seed solution. Activating a culture medium: 12 g/L glucose, 6 g/L peptone, 3 g/L potassium dihydrogen phosphate, 2 g/L disodium hydrogen phosphate, pH 6.5.

Preferred fermentation media are used: 16 g/L soluble starch, 15 g/L glycerin, 6 g/L tryptone, 4 g/L yeast powder, 7 g/L corn steep liquor and 3 g/L NH₄NO₃，0.8 g/L MnSO₄，2 g/L MgSO₄，2.5 g/L Na₂HPO₄，3 g/L K₂HPO₄8g/L colloidal chitin. Adding 70% volume of the culture medium into the fermentation medium, steam sterilizing at 118 deg.C for 20 min, inoculating activated strain at 6% (v/v), and controlling fermentationThe pH value in the process is maintained at 6.8, the culture temperature is adjusted to 29 ℃, the dissolved oxygen is maintained at more than 5 percent, the fermentation is completed after 4 days of fermentation, and the enzyme solution can be used after centrifugal filtration sterilization. After the fermentation, the chitinase activity was determined to be 36.39U/mL.

Example 5: preparation of chitin oligosaccharide by enzymolysis of substrate chitin

Centrifuging the fermented enzyme solution at 8000 rpm for 5min, collecting 100 mL of fermented supernatant, adding 10 g of chitin colloid, slowly oscillating in a constant-temperature water bath at 40 ℃ for 20 h, after the reaction is finished, adding absolute ethyl alcohol until the final volume fraction is 25% to precipitate the enzyme protein, centrifuging at 8000 rpm for 5min to remove the enzyme protein, then adding absolute ethyl alcohol until the final volume fraction is 80% to precipitate a chitooligosaccharide product, finally centrifuging at 10000 rpm for 5min to collect the chitooligosaccharide precipitate, and drying to obtain a formed chitooligosaccharide product of 7.12 g, wherein the actual product yield of the chitooligosaccharide is 71.2%.

Through determination, the initial enzyme solution is 38.07U/mL, the activity of residual chitinase is 20.32U/mL after the enzyme catalysis reaction is carried out for 20 hours, the half-life period of chitinase derived from chaetomium globosum HY1 in a reaction system is long, the enzyme is relatively stable, and the method has a relatively high industrial application value.

Claims (3)

1. A method for producing chitinase by fermentation of chaetomium globosum is characterized by comprising the following specific steps:

(1) strain: chaetomium globosumChaetomiumglobosum，The preservation name is: HY1, depository: china general microbiological culture Collection center (CGMCC) with the collection number of CGMCC No. 10609;

(2) activating strains: transferring the strain stored on the test tube slant into an activation culture medium, and activating at 28 deg.C and 200rpm until the strain is turbid to obtain activated seed solution; activating a culture medium: 8-15 g/L glucose, 3-6 g/L peptone, 1-3 g/L potassium dihydrogen phosphate, 1-2.5 g/L disodium hydrogen phosphate, and pH 6.5-7.0;

(3) fermentation production of chitinase

1) Fermentation medium: 15-20 g/L soluble starch, 10-15 g/L glycerin, 5-10 g/L tryptone, 2-4 g/L yeast powder,4-8 g/L corn steep liquor and 2-5 g/L NH₄NO₃，0.5～1.5 g/L MnSO₄，2～4 g/L MgSO₄，1～2.5 g/L Na₂HPO₄，1～3 g/L K₂HPO₄4-8 g/L colloidal chitin, and the pH value is 6.5-7.5;

2) shake cultivation or fermentation tank cultivation:

shake cultivation: inoculating the activated seed liquid into the sterilized fermentation medium according to the inoculation amount of 3-6% (v/v), culturing in a rotary shaking table at 200rpm, wherein the culture temperature is 28-30 ℃, after fermentation for 3-5 days, culturing is finished, and performing centrifugal filtration sterilization to obtain the enzyme liquid;

culturing in a fermentation tank: the method comprises the steps of filling 70% of the culture medium into a fermentation medium according to a volume ratio, sterilizing the culture medium for 20 min by steam at 118 ℃, inoculating activated strains according to a ratio of 3-6% (v/v), controlling the pH value in the fermentation process to be maintained at 6.5-7.5, adjusting the culture temperature to be 28-30 ℃, maintaining the dissolved oxygen at more than 5%, fermenting for 3-5 days, and performing centrifugal filtration sterilization to obtain the enzyme liquid.

2. A chitinase produced by the method of claim 1.

3. Use of the chitinase of claim 2 in the preparation of chitooligosaccharides.