CN111944789B - Method for producing chitinase by fermenting chaetomium globosum and application thereof - Google Patents
Method for producing chitinase by fermenting chaetomium globosum and application thereof Download PDFInfo
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- 102000012286 Chitinases Human genes 0.000 title claims abstract description 32
- 108010022172 Chitinases Proteins 0.000 title claims abstract description 32
- 241001515917 Chaetomium globosum Species 0.000 title claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 13
- 238000000855 fermentation Methods 0.000 claims abstract description 50
- 230000004151 fermentation Effects 0.000 claims abstract description 50
- 229920002101 Chitin Polymers 0.000 claims abstract description 29
- 239000001963 growth medium Substances 0.000 claims abstract description 19
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 18
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 7
- 239000001888 Peptone Substances 0.000 claims abstract description 7
- 108010080698 Peptones Proteins 0.000 claims abstract description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 7
- 229920002472 Starch Polymers 0.000 claims abstract description 7
- 240000008042 Zea mays Species 0.000 claims abstract description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 7
- 230000004913 activation Effects 0.000 claims abstract description 7
- 235000005822 corn Nutrition 0.000 claims abstract description 7
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims abstract description 7
- 239000008103 glucose Substances 0.000 claims abstract description 7
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 7
- 235000019319 peptone Nutrition 0.000 claims abstract description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 7
- 239000008107 starch Substances 0.000 claims abstract description 7
- 235000019698 starch Nutrition 0.000 claims abstract description 7
- 239000012137 tryptone Substances 0.000 claims abstract description 7
- 102000004190 Enzymes Human genes 0.000 claims description 22
- 108090000790 Enzymes Proteins 0.000 claims description 22
- 230000003213 activating effect Effects 0.000 claims description 14
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- 229920001661 Chitosan Polymers 0.000 claims description 6
- 235000011187 glycerol Nutrition 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 239000001301 oxygen Substances 0.000 claims description 4
- 229910052760 oxygen Inorganic materials 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 3
- 241000221955 Chaetomium Species 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims description 2
- 101710151413 Chitinase 1 Proteins 0.000 claims 1
- 102100037328 Chitotriosidase-1 Human genes 0.000 claims 1
- 101710132290 Chitotriosidase-1 Proteins 0.000 claims 1
- 101710107327 Endochitinase 1 Proteins 0.000 claims 1
- 238000009629 microbiological culture Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 14
- 229920001542 oligosaccharide Polymers 0.000 abstract description 5
- 150000002482 oligosaccharides Chemical class 0.000 abstract description 5
- 238000012136 culture method Methods 0.000 abstract description 4
- 235000016709 nutrition Nutrition 0.000 abstract description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 abstract 2
- 229910000397 disodium phosphate Inorganic materials 0.000 abstract 1
- 235000001727 glucose Nutrition 0.000 abstract 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 abstract 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 abstract 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000593 degrading effect Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000007515 enzymatic degradation Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 1
- 241000238421 Arthropoda Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 229940101006 anhydrous sodium sulfite Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000002144 chemical decomposition reaction Methods 0.000 description 1
- 239000011365 complex material Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000001476 sodium potassium tartrate Substances 0.000 description 1
- 235000011006 sodium potassium tartrate Nutrition 0.000 description 1
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2442—Chitinase (3.2.1.14)
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01014—Chitinase (3.2.1.14)
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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
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.
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