CN113234633A - Strain for producing chitinase and application thereof in preparation of chitooligosaccharide - Google Patents
Strain for producing chitinase and application thereof in preparation of chitooligosaccharide Download PDFInfo
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- CN113234633A CN113234633A CN202110588460.0A CN202110588460A CN113234633A CN 113234633 A CN113234633 A CN 113234633A CN 202110588460 A CN202110588460 A CN 202110588460A CN 113234633 A CN113234633 A CN 113234633A
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- 102000012286 Chitinases Human genes 0.000 title claims abstract description 75
- 108010022172 Chitinases Proteins 0.000 title claims abstract description 75
- 229920001661 Chitosan Polymers 0.000 title claims description 8
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 68
- 230000004151 fermentation Effects 0.000 claims abstract description 67
- 229920002101 Chitin Polymers 0.000 claims abstract description 56
- 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 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 15
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- 239000002609 medium Substances 0.000 claims description 19
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 15
- 229910052799 carbon Inorganic materials 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 10
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- 239000001301 oxygen Substances 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- QLTSDROPCWIKKY-PMCTYKHCSA-N beta-D-glucosaminyl-(1->4)-beta-D-glucosamine 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](CO)O1 QLTSDROPCWIKKY-PMCTYKHCSA-N 0.000 claims description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 7
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 6
- 230000002255 enzymatic effect Effects 0.000 claims description 6
- 239000007836 KH2PO4 Substances 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 5
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- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 3
- 241000192142 Proteobacteria Species 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000008101 lactose Substances 0.000 claims description 3
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- 235000019698 starch Nutrition 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 241000192001 Pediococcus Species 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 150000002314 glycerols Chemical class 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 239000000413 hydrolysate Substances 0.000 claims 2
- 241000588653 Neisseria Species 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 58
- 108090000790 Enzymes Proteins 0.000 abstract description 58
- 238000004519 manufacturing process Methods 0.000 abstract description 7
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- 239000000047 product Substances 0.000 description 23
- 230000000694 effects Effects 0.000 description 22
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- 239000007788 liquid Substances 0.000 description 10
- 235000000346 sugar Nutrition 0.000 description 10
- 239000000203 mixture Substances 0.000 description 9
- 229920001542 oligosaccharide Polymers 0.000 description 7
- 150000002482 oligosaccharides Chemical class 0.000 description 7
- 238000004809 thin layer chromatography Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 241000583256 Chitinibacter Species 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 238000010564 aerobic fermentation Methods 0.000 description 4
- 238000009776 industrial production Methods 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 238000010587 phase diagram Methods 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 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 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000238557 Decapoda Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 150000002772 monosaccharides Chemical class 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 150000004043 trisaccharides Chemical class 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 2
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 2
- 241000588656 Neisseriaceae Species 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
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- -1 chemical engineering Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 150000002016 disaccharides Chemical class 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
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- 238000001976 enzyme digestion Methods 0.000 description 2
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- 235000013376 functional food Nutrition 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229950006780 n-acetylglucosamine Drugs 0.000 description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 239000004753 textile Substances 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000472134 Chitinilyticum Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 238000002144 chemical decomposition reaction Methods 0.000 description 1
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- 108091036078 conserved sequence Proteins 0.000 description 1
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- ZRSNZINYAWTAHE-UHFFFAOYSA-N p-methoxybenzaldehyde Chemical compound COC1=CC=C(C=O)C=C1 ZRSNZINYAWTAHE-UHFFFAOYSA-N 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
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- 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
- 229940074439 potassium sodium tartrate Drugs 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 150000004044 tetrasaccharides Chemical class 0.000 description 1
- 238000005406 washing 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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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- 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 relates to a strain for producing chitinase and application thereof in preparation of chitosan oligosaccharide. The bacterial strain producing chitinase is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2021418 and the preservation date of 2021, 4 and 20 months. The invention also provides an application method for producing chitinase by using the bacterial strain for producing chitinase and preparing chitosan oligosaccharide by degrading chitin by using the chitinase. Compared with the prior art, the chitinase-producing strain has novel biological sources, simple fermentation method, lower enzyme production cost and high application value. In addition, the chitinase from the strain can be obtained in engineering bacteria through a clone table, and the large-scale preparation of the chitinase and the large-scale production of the chitosan oligosaccharide are expected to be realized.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a bacterial strain for producing chitinase and application thereof in preparation of chitosan oligosaccharide.
Background
Chitin (chitin), also known as chitin and chitin, is a linear polysaccharide made of N-acetyl-D-glucosamine (GlcNAC) linked by β -1, 4 glycosidic bonds. The storage amount is very rich, the content of the chitin in the nature is only second to that of cellulose, the chitin is the second most renewable resource in the nature except for cellulose, the annual biosynthesis amount of the chitin is far more than 100 hundred million tons, and the chitin is a huge treasury of renewable resources for human beings. Chitin is insoluble in water, organic solvents and alkali liquids, and therefore is difficult to be directly utilized. However, the chitosan oligosaccharide and chitosan obtained after the chitin is degraded or deacetylated have extremely high application value. The chitosan oligosaccharide has small molecular weight, is easy to absorb and utilize, and thus has better physiological activity, such as tumor resistance, immunity enhancement, blood sugar reduction, intestinal flora regulation, inflammation resistance and the like.
At present, the industrial production method of chitin oligosaccharide and chitosan oligosaccharide mainly uses shrimp and crab shells as raw materials, and calcium salt and protein are removed through the treatment of concentrated hydrochloric acid and sodium hydroxide, so that chitin is obtained. The chitin and chitosan are treated by a physical and chemical degradation method to obtain the chitosan oligosaccharide and chitosan oligosaccharide with lower polymerization degree. However, this method is expensive, causes a large environmental pollution, and has an uncontrollable degree of polymerization. The enzyme method is stable, has no pollution to the environment and lower cost, can obtain the chitooligosaccharide with fixed polymerization degree, and has huge application and development prospects.
At present, the best research is microbial chitinase, which is mainly to obtain excellent strains through natural screening or cloning expression, generate specific chitinase and degrade chitin to obtain specific chitin oligosaccharide products. Therefore, the chitin and the derivatives thereof have wider application, and lay a foundation for future industrial production and application.
The chitinases produced by different strains are different, and the enzyme-producing fermentation conditions of the strains are different, so that the screening of the strains which efficiently produce the chitinases and the study of the enzyme-producing fermentation conditions of the strains are also important in the study of chitin. With the continuous and deep research on chitinase, the industrial production of chitin and derivatives thereof is mature, gradually scaled and industrialized.
Although chitosan oligosaccharide has been widely applied to the fields of medicine, chemical engineering, textile, printing and dyeing, paper making, functional food, environmental protection and the like after long-term research, the production process is still not mature, and the industrial production condition is still not perfect. Therefore, further research and development of new chitinase are needed to lay a foundation for the production of chitin oligosaccharide.
Disclosure of Invention
The invention aims to provide a bacterial strain for producing chitinase and application thereof in preparation of chitosan oligosaccharide.
The invention screens and obtains a strain for producing chitinase from the bottom soil of a shrimp and crab farm, and the invention also uses the strain for producing chitinase as an initial strain to explore a specific fermentation method for producing chitinase by researching physicochemical properties and optimizing fermentation conditions so as to obtain a specific chitin oligosaccharide product. The prepared enzyme can hydrolyze chitin to generate chitosan oligosaccharide, and the chitosan oligosaccharide generated by enzymolysis can be widely applied to the fields of medicine, chemical industry, printing and dyeing, papermaking, textile, functional food, environmental protection and the like.
The purpose of the invention can be realized by the following technical scheme:
the invention firstly provides a strain for producing chitinase, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2021418 and the preservation date of 2021 year, 4 months and 20.
The chitinase-producing strain can produce chitinase system and degrade chitin, and the main products are chitin monosaccharide, disaccharide and trisaccharide.
The chitinase-producing strain is determined to be a new strain of the genus chitinibacillus of the class Proteobacteria, the family Neisseriaceae and the name chitinibacter sp.ZQ-8 through colony morphology observation and molecular identification.
The study on the morphology and the physicochemical property of the strain producing the chitinase is characterized in that bacterial colonies are wet, smooth, transparent and easy to pick, the texture is uniform, the color of the edge is consistent with that of the center, and the periphery of the bacterial colonies can be observed to be obviously transparent on a solid screening culture medium.
In one embodiment of the invention, the 16S rRNA gene of the chitinase-producing strain is shown in SEQ ID No. 1.
The invention also provides a fermentation method of the chitinase-producing strain, which comprises the following steps: inoculating the strain producing chitinase into a fermentation culture medium to serve as seed liquid for culture, and inoculating the shaken seed liquid into the fermentation culture medium for culture.
In one embodiment of the present invention, when the chitinase-producing strain is inoculated into a fermentation medium to be cultured as a seed solution, the chitinase-producing strain is present in the form of a glycerol strain or a pediococcus strain.
In one embodiment of the present invention, when the chitinase-producing strain is inoculated into a fermentation medium to be cultured as a seed solution, the culture conditions are as follows: incubated at 37 ℃ for 1 day.
In one embodiment of the present invention, the shaken seed solution is inoculated into a fermentation medium and cultured under the following conditions: the inoculum size was 1%, and the cells were cultured at 37 ℃ under 200prm conditions for 3 days.
In one embodiment of the invention, the composition of the fermentation medium is:
fermentation medium (g/L): 10-30 parts of carbon source, 5-15 parts of peptone, 2.5-5.0 parts of NaCl and KH2PO4 0.3～1.2，K2HPO4 0.1～0.5，MgSO4 0.5～1.0，pH7.0～8.0；
1-3 per mill of colloidal chitin is also added into the fermentation medium as an inducer;
the carbon source is selected from one or more of chitin powder, colloidal chitin, glucose, fructose, sucrose, starch, lactose, cellulose powder or xylose, and is preferably chitin powder.
In one embodiment of the present invention, preferably, the composition of the fermentation medium is:
fermentation medium (g/L): carbon source 30, peptone 10, NaCl 2.5, KH2PO4 0.7，K2HPO4 0.3，MgSO40.5, pH7.0; 1 per mill of colloidal chitin is also added into the fermentation medium as an inducer.
Preferably, the carbon source is chitin powder.
In one embodiment of the present invention, the fermentation culture of the chitinase-producing strain is carried out in the presence of oxygen.
Experimental studies show that the chitinase-producing strain is facultative anaerobe and can produce more chitinase under the condition of sufficient oxygen.
The invention also provides application of the bacterial strain for producing the chitinase, the chitinase is produced by utilizing the bacterial strain for producing the chitinase, and the chitinase is used for degrading chitin to prepare the chitooligosaccharide.
In one embodiment of the present invention, after fermentation, the chitinase-producing strain is subjected to simple pretreatment to obtain a crude enzyme solution, and a colloidal chitin is added to react for a period of time to detect the product, so that the study finds that chitotriose, tetrasaccharide and oligosaccharide with even higher polymerization degree can be produced in a short time, and finally, chitobiose, trisaccharide and partial monosaccharide are mainly remained.
In one embodiment of the present invention, different concentrations of chitooligosaccharides are obtained by varying the fermentation conditions or enzymatic hydrolysis conditions.
The chitinase-producing strain produces one or more chitinases by fermentation. By changing the fermentation conditions, the yield and the proportion of different chitinases can be controlled. Meanwhile, the chitin oligosaccharide with different concentrations can be obtained by changing the enzymolysis conditions. Under the condition of no oxygen or less oxygen, the thalli grow slowly, and the crude enzyme enzymolysis product produced by fermentation is mainly chitobiose. Under the condition of sufficient oxygen, the thallus grows faster, and the crude enzyme enzymolysis products produced by fermentation mainly comprise chitobiose and chitotriose.
Based on the above researches, the crude enzyme enzymolysis products produced by the flat-bottom shake flask fermentation of the invention are mainly chitobiose, and the crude enzyme enzymolysis products produced by the baffle flask fermentation are mainly chitobiose and chitotriose.
In one embodiment of the invention, the chitinase derived from the chitinase-producing strain can be obtained from cloning expression engineering bacteria, and the chitinase is expected to be prepared in a large amount and used for large-scale production of chitosan oligosaccharide.
Compared with the prior art, the beneficial effects of the invention are embodied in the following aspects:
the strain for producing the chitinase has novel biological sources, simple fermentation method, lower enzyme production cost and high application value. In addition, the chitinase from the strain can be obtained in engineering bacteria through a clone table, and the large-scale preparation of the chitinase and the large-scale production of the chitosan oligosaccharide are expected to be realized.
Drawings
FIG. 1 is a photograph showing the colony morphology of Chitiniyticum sp.ZQ-8 strain.
FIG. 2 shows a phylogenetic tree constructed based on the 16SrRNA gene sequence.
FIG. 3 shows the effect of different carbon sources on enzyme activity.
FIG. 4 shows the results of TLC analysis of the enzymatic products. No.1 and No. 2 are enzymolysis products, and M is N-acetylglucosamine.
FIG. 5 is a TLC diagram of products of No.1 and No. 2 enzymes at different enzymolysis temperatures.
FIG. 6 is a liquid phase diagram of the product of No.1 enzyme at 50 ℃.
FIG. 7 is a liquid phase diagram of the product of No. 2 enzyme digestion at 50 ℃.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
Separation and identification of chitinase-producing strain Chitinilyticum sp.ZQ-8
A strain for producing chitinase is obtained by screening from the soil at the bottom of a shrimp and crab culturing farm, and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2021418.
The chitinase-producing strain is determined to be a new strain of the genus chitinibacillus of the class Proteobacteria, the family Neisseriaceae and the name chitinibacter sp.ZQ-8 through colony morphology observation and molecular identification.
1) Morphological identification
The Chitiniltyticum sp.ZQ-8 strain is inoculated into a solid screening culture medium by a streak method for culture, and the colony morphology is observed after 2 days. The results show that the bacterial colony is wet, smoother, more transparent, easy to pick, uniform in texture, consistent in color of the edge and the center, and preliminarily judged to be bacteria. The bacteria were determined to be gram negative by gram staining. The colony morphology is shown in figure 1.
2) Molecular identification
The conserved sequence can be directly cloned by PCR by using a conventional 16SrRNA sequencing method because the bacterium is a gram-negative bacterium, the primers are 27F:5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R:5'-GGTTACCTTGTTACGACTT-3', and the cloned sequence is sent to a biological company for sequencing. The 16S rRNA gene of the chitinase-producing strain is shown as SEQ ID No. 1. Comparing the sequencing result with the existing sequence, finding that the sequence is closest to the chitinibacter litopenaei C1, and presuming that the strain belongs to a new strain of the chitinibacter by establishing a phylogenetic tree (as shown in figure 2) and analyzing according to the genetic relationship.
Example 2
Chitinase producing strain chitinilinyticum sp.ZQ-8 crude enzyme activity determination
Preparing colloidal chitin:
(1) precooled 300ml concentrated hydrochloric acid was slowly added to a beaker containing 10g chitin powder and stirred well.
(2) 100ml of distilled water was added and stirred to form a paste, which was placed in a refrigerator at 4 ℃ overnight.
(3) After complete swelling, adding distilled water and stirring until the solution turns milky white.
(4) Centrifuging at 6000r/min for 10min, discarding supernatant and washing precipitate with water. This was repeated until the solution was neutral.
(5) Finally, the volume is fixed to 1000ml, and the 1 percent colloidal chitin is obtained.
DNS configuration:
3.15g of 3, 5-dinitrosalicylic acid is taken and added into 500ml of water, the water bath is carried out to 45 ℃, the stirring is carried out for 5s, then 20g of NaOH is slowly added, and the stirring is carried out continuously until the mixture is transparent. And adding 91g of potassium sodium tartrate, 2.5g of phenol and 2.5g of anhydrous sodium nitrite, continuously heating, supplementing water to 300ml, stirring until the water is dissolved, cooling, fixing the volume to 1000ml, filtering and storing in the dark place. Can be used after one week, and has effective period of 6 months.
And (3) enzyme activity determination:
1) pretreatment of fermentation liquor: centrifuging the fermentation liquid at 8000r/min for 10min, and collecting the supernatant.
2) Mu.l of the crude enzyme solution was mixed with 400. mu.l of 1% colloidal chitin, and subjected to a water bath at 50 ℃ for 10 min.
3) Then 500. mu.l DNS was added, 1000. mu.l water was added to the mixture to make 2ml, and OD was measured at 540nm after 10min in boiling water bath.
Definition of enzyme activity: under the optimal conditions, the mu mol number of reducing sugar generated by degrading chitin in 1mL of enzyme solution for 1min is defined as one enzyme activity unit U.
Example 3
Fermentation condition optimization of chitinase producing bacteria Chitininyticum sp.ZQ-8
Basal fermentation medium (g/L): colloidal chitin 10, peptone 10, NaCl 5, KH2PO4 0.7，K2HPO40.3，MgSO40.5, pH 7.0. The enzyme activity of the medium after 3 days of fermentation at 37 ℃ in a flat-bottomed flask was 0.20U/ml.
Influence of dissolved oxygen on conditions of enzyme production by fermentation
The experiments can be divided into 5 groups according to the difference of dissolved oxygen, wherein the first group is subjected to sealed anaerobic fermentation by using 50ml of fermentation liquid in a 250ml flat-bottom conical bottle, the second group is subjected to aerobic fermentation by using 50ml of fermentation liquid in a 250ml flat-bottom conical bottle, the third group is subjected to aerobic fermentation by using 30ml of fermentation liquid in a 250ml flat-bottom conical bottle, the fourth group is subjected to aerobic fermentation by using 50ml of fermentation liquid in a 250ml baffle conical bottle, and the fifth group is subjected to aerobic fermentation by using 30ml of fermentation liquid in a 250ml baffle conical bottle. The dissolved oxygen of the first group to the fifth group is gradually increased. The five groups of shake flasks were fermented at 37 ℃ for three days, and the enzyme activities were measured.
The result shows that the enzyme activity of the first group is 0.12U/ml, the enzyme activity of the second group is 0.20U/ml, the enzyme activity of the third group is 0.42U/ml, the enzyme activity of the fourth group is 1.26U/ml, and the enzyme activity of the fifth group is 1.64U/ml, wherein the enzyme activity of the fifth group is the highest, and compared with a basic fermentation culture medium, the enzyme activity is improved by about 8 times. The bacterium is more beneficial to producing chitinase under the condition of sufficient oxygen.
Influence of the amount of carbon Source and carbon Source added
Powdery chitin, glucose, fructose, sucrose, starch, lactose, cellulose powder, xylose and the like are selected as carbon sources to replace colloidal chitin on a basic fermentation medium, the addition amount of the carbon sources is 1 percent, namely 10g/L, 1 per mill of colloidal chitin is added as an inducer, and the mixture is fermented for 3 days at 37 ℃ by a baffle cone bottle.
The results are shown in FIG. 3. As shown in FIG. 3, the powdered chitin has the best effect as a carbon source, and the enzyme activity of the fermented crude enzyme is 1.82U/ml. The chitin powder has low preparation cost and wide source, and is a relatively suitable carbon source.
Under the condition of keeping other conditions unchanged, the addition amount of the carbon source (chitin powder) is changed, and fermentation is carried out under the same conditions. The result shows that the enzyme activity is highest when 3 percent of chitin powder is added, and the enzyme activity can reach 2.24U/ml. Therefore, the appropriate addition of carbon source in the fermentation culture is beneficial to improving the enzyme activity.
Example 4: preparation and detection of chitin oligosaccharide
Preparation of crude enzyme solution: firstly, glycerol bacteria or flat bacteria are inoculated into a fermentation culture medium for activation for 1 day, then 1 percent of seed liquid is inoculated into the fermentation culture medium, the culture is carried out for three days at 37 ℃ and 200rpm, and the composition of the fermentation culture medium (g/L) is as follows: chitin powder 30, peptone 10, NaCl 2.5, KH2PO4 0.7，K2HPO4 0.3，MgSO40.5, pH7.0, and adding 1 per mill of colloidal chitin as an inducer. The obtained fermentation liquor is centrifuged at 8000rpm for 10min, and then the supernatant is collected. 10U of the crude enzyme solution is taken, 100mL of 1% colloidal chitin is added, and the mixture reacts in a water bath kettle at the constant temperature of 50 ℃ for 24 hours while stirring. After the reaction is finished, centrifuging the mixture for 10 minutes at 10000rpm, removing residual impurities and unreacted substrates, and collecting supernatant for identification.
TLC identification: firstly, drawing a straight line at the position 1cm away from the top end of a thin-layer chromatography plate, and sampling every 1cm, wherein 3 mu l of enzymolysis product is sampled. And (5) putting the spotted sample into an unfolding cylinder for unfolding. The developing agent formula (v/v) is isopropanol: water: ammonia water 15: 1: 7.5. the developer formula (v/v) is anisic aldehyde: ethanol: concentrated sulfuric acid: acetic acid 5: 90: 5: 1.
the results of the identification are shown in FIG. 4. The results show that the final product is chitin monosaccharide, disaccharide and trisaccharide.
Example 5: analysis of enzymatic products under different conditions
In this example, two groups of enzymes were obtained by flat-bottom shake flask and baffle-shake flask fermentations, and the fermentation media and fermentation methods were the same as those in example 4. The number of the crude enzyme produced by flat-bottom shake flask fermentation is 1, the number of the crude enzyme produced by baffle shake flask fermentation is 2, and the fermentation conditions of the two groups of enzymes are consistent except different shake flasks. The enzyme activities and products of the reaction of the No.1 enzyme and the No. 2 enzyme at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃ are respectively measured.
The results of enzyme activity are shown in Table 1, which revealed that the optimum reaction temperatures for enzyme No.1 and enzyme No. 2 were both 50 ℃.
TABLE 1 enzymatic Activity at different enzymatic temperatures
Then, the substrate is subjected to enzymolysis by using No.1 enzyme and No. 2 enzyme, and products and yield of the substrate at different temperatures are measured. The specific method comprises the following steps:
taking 2ml of 1% colloidal chitin, namely 20mg of substrate, adding 200ul of No.1 or No. 2 crude enzyme solution, performing enzymolysis at 30 ℃, 40 ℃, 50 ℃ and 60 ℃ for 24h respectively, detecting the composition of the product by TLC, and detecting the content of each component by HPLC.
TLC results showed: the enzyme No.1 is mainly used for producing the chitobiose under different temperature conditions. The No. 2 enzyme can produce two or three sugars mainly under the condition of 50 ℃. The contents of sugars after enzymolysis at 50 ℃ were quantitatively determined by HPLC using enzyme No.1 and enzyme No. 2, and the results are shown in tables 2 and 3.
TABLE 21 enzyme hydrolysis at 50 deg.C for 24h each sugar content
TABLE 32 enzyme 50 ℃ for 24h each sugar content
Degradation rate is the mass of each sugar/total mass of substrate after degradation
The ratio of each sugar to the total sugar content is 100%
Each sugar concentration is (standard concentration x sample peak area)/standard peak area;
wherein, the standard product refers to a pure chitosan oligosaccharide standard product, and the sample refers to an enzymolysis sample obtained through experiments.
Therefore, the yield and the yield are different under different conditions, so the yield and the proportion of the chitosan oligosaccharide can be controlled by controlling the enzymolysis temperature and the fermentation conditions.
FIG. 5 is a TLC diagram of products of No.1 and No. 2 enzymes at different enzymolysis temperatures.
FIG. 6 is a liquid phase diagram of the product of No.1 enzyme at 50 ℃.
FIG. 7 is a liquid phase diagram of the product of No. 2 enzyme digestion at 50 ℃.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A bacterial strain for producing chitinase is characterized in that the bacterial strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2021418 and the preservation date of 2021 year, 4 months and 20.
2. The chitinase-producing strain of claim 1, wherein the chitinase-producing strain is a novel strain of the genus chitinicum of the family Neisseria of the class Proteobacteria.
3. The chitinase-producing strain according to claim 1, wherein the 16S rRNA gene of the chitinase-producing strain is shown in SEQ ID No. 1.
4. A fermentation method of the chitinase-producing strain according to claim 1, wherein the chitinase-producing strain is inoculated into a fermentation medium as a seed solution, cultured, and then inoculated into the fermentation medium for culture.
5. The fermentation method of the chitinase-producing strain according to claim 4, wherein the chitinase-producing strain is present in the form of a glycerol bacterium or a pediococcus strain when the chitinase-producing strain is inoculated into a fermentation medium to be cultured as a seed solution.
6. The fermentation method of the chitinase-producing strain according to claim 4, wherein when the chitinase-producing strain is inoculated into a fermentation medium to be cultured as a seed solution, the culture conditions are as follows: culturing at 37 deg.C for 1 day, inoculating the shaken seed solution into fermentation culture medium under the following conditions: the inoculum size was 1%, and the cells were cultured at 37 ℃ under 200prm conditions for 3 days.
7. The method of fermenting a chitinase-producing strain according to claim 4, wherein the fermentation medium consists of:
fermentation medium (g/L): 10-30 parts of carbon source, 5-15 parts of peptone, 2.5-5.0 parts of NaCl and KH2PO4 0.3～1.2，K2HPO40.1～0.5，MgSO4 0.5～1.0，pH7.0～8.0；
1-3 per mill of colloidal chitin is also added into the fermentation medium as an inducer;
the carbon source is selected from one or more of chitin powder, colloidal chitin, glucose, fructose, sucrose, starch, lactose, cellulose powder or xylose.
8. The method of claim 4, wherein the fermentation of the chitinase-producing strain is carried out in the presence of oxygen.
9. The application of the strain for producing the chitinase is characterized in that the chitinase is produced by using the strain for producing the chitinase, and the chitinase is used for degrading chitin to prepare the chitooligosaccharide.
10. The use of the chitinase-producing strain of claim 9, wherein the crude enzymatic hydrolysate produced by fermentation in a flat-bottom shake flask is mainly chitobiose, and the crude enzymatic hydrolysate produced by fermentation in a baffle-flask is mainly chitobiose and chitotriose.
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| CN102286414A (en) * | 2011-09-02 | 2011-12-21 | 海南大学 | Chitin-degrading bacterial strain and method for preparing chitooligosaccharide by utilizing same |
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| US20210002684A1 (en) * | 2018-11-19 | 2021-01-07 | Changshu Institute Of Technology | Strain for producing chitinase and application thereof |
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| CN102286414A (en) * | 2011-09-02 | 2011-12-21 | 海南大学 | Chitin-degrading bacterial strain and method for preparing chitooligosaccharide by utilizing same |
| CN104130961A (en) * | 2014-07-25 | 2014-11-05 | 南京工业大学 | Bacterial strain for producing chitinase and its use in chitin enzymolysis |
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