CN113234633B - Strain for producing chitinase and application of strain in preparation of chitosan oligosaccharide - Google Patents
Strain for producing chitinase and application of strain in preparation of chitosan oligosaccharide Download PDFInfo
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- 102000012286 Chitinases Human genes 0.000 title claims abstract description 59
- 108010022172 Chitinases Proteins 0.000 title claims abstract description 59
- 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 title abstract description 23
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 64
- 108090000790 Enzymes Proteins 0.000 claims abstract description 64
- 238000000855 fermentation Methods 0.000 claims abstract description 58
- 230000004151 fermentation Effects 0.000 claims abstract description 57
- 229920002101 Chitin Polymers 0.000 claims abstract description 42
- 229920001661 Chitosan Polymers 0.000 claims abstract description 19
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 230000000593 degrading effect Effects 0.000 claims abstract description 5
- 241000472134 Chitinilyticum Species 0.000 claims description 12
- 230000002255 enzymatic effect Effects 0.000 claims description 4
- 241000588656 Neisseriaceae Species 0.000 claims description 3
- 239000000413 hydrolysate Substances 0.000 claims 2
- 241000894006 Bacteria Species 0.000 abstract description 12
- 238000000034 method Methods 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 239000000047 product Substances 0.000 description 23
- 230000000694 effects Effects 0.000 description 22
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 13
- 229910052799 carbon Inorganic materials 0.000 description 13
- 239000002609 medium Substances 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 239000007788 liquid Substances 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 8
- 229910052760 oxygen Inorganic materials 0.000 description 8
- 239000001301 oxygen Substances 0.000 description 8
- 230000007071 enzymatic hydrolysis Effects 0.000 description 7
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 7
- 238000004809 thin layer chromatography Methods 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 239000000084 colloidal system Substances 0.000 description 5
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 238000010564 aerobic fermentation Methods 0.000 description 4
- 239000000411 inducer Substances 0.000 description 4
- 238000009776 industrial production Methods 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 238000010587 phase diagram Methods 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000012163 sequencing technique 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
- 238000006243 chemical reaction Methods 0.000 description 3
- 150000002016 disaccharides Chemical class 0.000 description 3
- 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 3
- 229940074439 potassium sodium tartrate Drugs 0.000 description 3
- 235000010288 sodium nitrite Nutrition 0.000 description 3
- 235000011006 sodium potassium tartrate Nutrition 0.000 description 3
- 150000004043 trisaccharides Chemical class 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- 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 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 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 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
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 241000588769 Proteus <enterobacteria> Species 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 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 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000006731 degradation reaction Methods 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
- 230000007613 environmental effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013376 functional food Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000008101 lactose Substances 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
- 238000005457 optimization Methods 0.000 description 2
- ZRSNZINYAWTAHE-UHFFFAOYSA-N p-methoxybenzaldehyde Chemical compound COC1=CC=C(C=O)C=C1 ZRSNZINYAWTAHE-UHFFFAOYSA-N 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000004753 textile Substances 0.000 description 2
- 241000274485 Chitinilyticum litopenaei Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 241000282414 Homo sapiens Species 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
- 241001052560 Thallis Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 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 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
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229940066779 peptones Drugs 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- -1 printing and dyeing Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- 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
- C12P19/26—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 relates to a strain for producing chitinase and application thereof in preparation of chitosan oligosaccharide. The 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 months and 20. The invention also provides an application method for producing chitinase by utilizing the strain for producing chitinase, and the chitinase is used for degrading chitin to prepare chitooligosaccharide. Compared with the prior art, the strain for producing the chitinase is novel in biological source, simple in fermentation method, low in enzyme production cost and high in application value. In addition, the chitinase from the strain can be cloned and expressed into engineering bacteria, so that the chitinase is expected to be prepared in a large scale and used for producing chitosan oligosaccharide in a large scale.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a strain for producing chitinase and application of the strain in preparation of chitosan oligosaccharide.
Background
Chitin (chitin), also known as chitin, is a linear polysaccharide formed by connecting N-acetyl-D-glucosamine (GlcNAC) through beta-1, 4 glycosidic bonds. The storage is very rich, the content in the nature is inferior to cellulose, the second biomass renewable resource except cellulose in the nature, the biosynthesis amount of chitin per year is far more than 100 hundred million tons, and the biomass renewable resource is a huge renewable resource treasury for human beings. Chitin is insoluble in water, organic solvents and lyes and is therefore more difficult to directly use. But the chitosan oligosaccharide and chitosan obtained after chitin is degraded or deacetylated have extremely high application value. The chitosan oligosaccharide has small molecular weight and is easier to absorb and utilize, so that the chitosan oligosaccharide has better physiological activity, such as anti-tumor, immunity enhancing, blood sugar reducing, intestinal flora regulating, anti-inflammatory and the like.
The current industrial production method of chitosan oligosaccharide and chitosan oligosaccharide mainly uses shrimp and crab shells as raw materials, and calcium salt and protein are removed by treatment of concentrated hydrochloric acid and sodium hydroxide to obtain chitin. Chitin and chitosan are treated by a physical and chemical degradation method, and chitosan oligosaccharide with low polymerization degree can be obtained. However, this method is costly, causes a large environmental pollution, and does not control the polymerization degree. The enzyme method is stable, has no pollution to the environment and lower cost, can obtain the chitosan oligosaccharide with fixed polymerization degree, and has huge application and development prospects.
The most studied microbial chitinase is mainly to obtain excellent strains through natural screening or cloning expression, generate specific chitinase and degrade chitin to obtain specific chitooligosaccharide products. Therefore, the chitin and the derivatives thereof can be widely applied, and a foundation is laid for the future industrial production and application.
Chitinase produced by different strains is different, and enzyme-producing fermentation conditions are different, so that screening out the strain capable of producing chitinase efficiently and researching the enzyme-producing fermentation conditions are also important in the aspect of researching chitin. Along with the continuous and deep research on chitinase, the industrial production of chitin and derivatives thereof is more and more mature, and the scale and industrialization are gradually realized.
Although the chitosan oligosaccharide has been widely used in the fields of medicine, chemical industry, textile, printing and dyeing, paper making, functional food, environmental protection and the like through long-time researches, 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, and a foundation is laid for the production of chitosan oligosaccharide.
Disclosure of Invention
The invention aims to provide a strain for producing chitinase and application thereof in preparation of chitosan oligosaccharide.
The invention screens out a strain producing chitinase from pool bottom soil of a shrimp and crab farm, and explores a specific fermentation method for producing chitinase by taking the strain producing chitinase as an initial strain through physical and chemical property research and fermentation condition optimization, thereby obtaining a specific chitooligosaccharide 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 aim of the invention can be achieved 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 to 4 months 20.
The strain producing chitinase can produce chitinase series and is used for degrading chitin, and main products are chitinase, disaccharide and trisaccharide.
The strain producing chitinase is identified by colony morphology observation and molecular identification, and is determined to be a new strain of the genus Chitinilyticum of the class Proteus, neisseriaceae, and named as Chitinilyticum sp.ZQ-8.
The strain morphology and physicochemical property research of the chitinase-producing strain is characterized in that the colony is moist, smoother, more transparent, easy to pick, uniform in texture, consistent in edge and central color, and obviously transparent around the colony can be observed 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 strain for producing chitinase, which comprises the following steps: inoculating the strain producing the chitinase into a fermentation culture medium to be used as seed liquid for culture, and inoculating the shaken seed liquid into the fermentation culture medium to be cultured.
In one embodiment of the present invention, the chitinase-producing strain is present in the form of glycerol bacteria or flat bacteria when the chitinase-producing strain is inoculated into a fermentation medium for cultivation as a seed liquid.
In one embodiment of the present invention, when the chitinase-producing strain is inoculated into a fermentation medium as a seed liquid for cultivation, the cultivation conditions are as follows: the culture was carried out at 37℃for 1 day.
In one embodiment of the present invention, the shaking seed liquid is inoculated into a fermentation medium under the following conditions: the inoculum size was 1%, and the cells were cultured at 37℃for 3 days under 200prm conditions.
In one embodiment of the invention, the fermentation medium has a composition of:
fermentation medium (g/L): 10 to 30 carbon sources, 5 to 15 peptones, 2.5 to 5.0 NaCl and KH 2 PO 4 0.3～1.2，K 2 HPO 4 0.1～0.5，MgSO 4 0.5～1.0，pH7.0～8.0；
Colloid chitin of 1-3 permillage is also added into the fermentation culture medium as inducer;
the carbon source is selected from one or more of chitin powder, colloid chitin, glucose, fructose, sucrose, starch, lactose, cellulose powder or xylose, etc., 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, KH 2 PO 4 0.7，K 2 HPO 4 0.3，MgSO 4 0.5, pH7.0; colloidal chitin of 1%is also added into the fermentation culture medium as an inducer.
Preferably, the carbon source is chitin powder.
In one embodiment of the invention, the fermentation culture of the chitinase-producing strain is performed in the presence of oxygen.
Experimental research shows that the strain for producing the chitinase is facultative anaerobe, and can produce more chitinase under the condition of sufficient oxygen.
The invention also provides an application of the strain for producing chitinase, which is used for producing chitinase and degrading chitin to prepare chitooligosaccharide.
In one embodiment of the invention, after fermentation of the chitinase-producing strain, a crude enzyme solution is obtained by simple pretreatment, and products are detected after colloidal chitin is added for a period of time, and it is found that chitosan, tetraose and oligosaccharides with even higher polymerization degree can be produced in a short time, and finally mainly chitosan, trisaccharide and partial monosaccharides remain.
In one embodiment of the invention, different concentrations of chitosan oligosaccharide are obtained by varying the fermentation or enzymatic conditions.
The chitinase-producing strain produces one or more chitinases by fermentation. By changing the fermentation conditions, the yield and the proportion of different chitinase can be controlled. Meanwhile, different concentrations of chitosan oligosaccharide can be obtained by changing enzymolysis conditions. Under the condition of no or little oxygen, the thallus grows slowly, and the crude enzyme enzymolysis product produced by fermentation is mainly chitosan. Under the condition of sufficient oxygen, the thalli grow faster, and the crude enzyme enzymolysis products generated by fermentation are mainly chitosan and chitosan.
Based on the research, the invention discovers that the crude enzyme enzymolysis product generated by flat bottom shake flask fermentation is mainly chitosan, and the crude enzyme enzymolysis product generated by baffle shake flask fermentation is mainly chitosan and chitosan.
In one embodiment of the invention, chitinase derived from the strain for producing chitinase can be expressed into engineering bacteria through cloning, so that mass production of chitinase and large-scale production of chitooligosaccharides are expected to be realized.
Compared with the prior art, the invention has the beneficial effects that:
the strain for producing the chitinase is novel in biological source, simple in fermentation method, low in enzyme production cost and high in application value. In addition, the chitinase from the strain can be cloned and expressed into engineering bacteria, so that the chitinase is expected to be prepared in a large scale and used for producing chitosan oligosaccharide in a large scale.
Drawings
FIG. 1 is a photograph of colony morphology of Chitinilyticum sp.ZQ-8 strain.
FIG. 2 is a phylogenetic tree constructed based on the 16SrRNA gene sequence.
FIG. 3 shows the effect of different kinds of carbon sources on enzyme activity.
FIG. 4 shows the results of analysis of the enzymatic hydrolysis products by TLC. No.1, 2 is the enzymatic hydrolysis product, M is N-acetylglucosamine.
FIG. 5 shows TLC patterns of products of enzyme No.1 and enzyme No. 2 at different enzymolysis temperatures.
FIG. 6 is a liquid phase diagram of the No.1 enzyme enzymatic hydrolysis product at 50 ℃.
FIG. 7 is a liquid phase diagram of the No. 2 enzyme enzymatic hydrolysis product at 50 ℃.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
Example 1
Isolation and characterization of chitinase-producing Strain Chitinilyticum sp.ZQ-8
The strain producing chitinase is obtained by screening from the pool bottom soil of a shrimp and crab culturing farm and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2021418.
The strain producing chitinase is identified by colony morphology observation and molecular identification, and is determined to be a new strain of the genus Chitinilyticum of the class Proteus, neisseriaceae, and named as Chitinilyticum sp.ZQ-8.
1) Morphological identification
The Chitinilyticum sp.ZQ-8 strain was inoculated into a solid screening medium by streaking for cultivation, and colony morphology was observed after 2 days. The results show that the bacterial colony is moist, smoother, more transparent, easy to pick, uniform in texture, consistent in color with the center at the edge, and primarily judges that the bacteria are bacteria. The bacteria were determined to be gram negative by gram staining. The colony morphology is shown in figure 1.
2) Molecular characterization
By adopting a conventional 16SrRNA sequencing method, the strain is gram negative, so that a conserved sequence can be cloned by direct PCR, the primers are 27F:5 '-AGAGTTTGATCCTGGGCTCGCTTAG-3', 1490 2R:5'-GGTTACCTTGTTACGACTT-3', and the cloned sequence is sent to a biological company for sequencing. The 16S rRNA gene of the strain producing the chitinase is shown as SEQ ID No. 1. Comparing the sequencing result with the existing sequence, finding that the sequencing result is closest to Chitinilyticum litopenaei C1, and presuming that the bacterium belongs to a new strain of Chitinilyticum by establishing a phylogenetic tree (shown in figure 2) and analyzing according to the genetic relationship.
Example 2
Enzyme activity determination of chitinase-producing bacterium Chitinilyticum sp.ZQ-8 crude enzyme
Colloid chitin configuration:
(1) Pre-chilled 300ml of concentrated hydrochloric acid was slowly added to a beaker containing 10g chitin powder with sufficient agitation.
(2) 100ml of distilled water was added and stirred into a paste, and put in a refrigerator at 4℃overnight.
(3) After complete swelling, distilled water was added and the solution was stirred until it became milky white.
(4) Centrifuging at 6000r/min for 10min, discarding supernatant, and washing the precipitate with water. This is repeated until the wash is neutral.
(5) Finally, the volume is fixed to 1000ml, and 1% of 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 mixture is stirred for 5s, then 20g of NaOH is slowly added, and the mixture is continuously stirred until the mixture is transparent. 91g of potassium sodium tartrate, 2.5g of phenol and 2.5g of anhydrous sodium nitrite are added, heating is continued, water is added to 300ml, stirring is carried out until the potassium sodium tartrate and the sodium nitrite are dissolved, cooling is carried out, the volume is fixed to 1000ml, and the potassium sodium tartrate and the sodium nitrite are filtered and stored in a dark place. Can be used after one week, and has a valid period of 6 months.
Enzyme activity determination:
1) Pretreatment of fermentation liquor: centrifuging the fermentation broth at 8000r/min for 10min, and collecting supernatant.
2) 100 μl of crude enzyme solution was mixed with 400 μl of 1% colloidal chitin, and the mixture was subjected to a water bath at 50deg.C for 10min.
3) Then, 500. Mu.l of DNS was added thereto, 1000. Mu.l of water was set to 2ml, and the OD was measured at 540nm after 10 minutes of boiling water bath.
Definition of enzyme activity: under the optimal conditions, the μmol number of reducing sugar generated by degrading chitin with 1mL of enzyme solution for 1min is defined as one enzyme activity unit U.
Example 3
Chitinase producing bacterium Chitinilyticum sp.ZQ-8 fermentation condition optimization
Basal fermentation Medium (g/L): colloidal chitin 10, peptone 10, naCl 5, KH 2 PO 4 0.7，K 2 HPO 4 0.3，MgSO 4 0.5, pH7.0. The enzyme activity of the medium after 3 days of fermentation at 37℃was 0.20U/ml using a flat-bottomed conical flask.
Influence of dissolved oxygen on the enzyme production of fermentation
The experiments can be divided into 5 groups according to dissolved oxygen, wherein the first group uses 50ml fermentation liquid in a 250ml flat-bottom conical bottle to perform sealed anaerobic fermentation, the second group uses 50ml fermentation liquid in a 250ml flat-bottom conical bottle to perform aerobic fermentation, the third group uses 30ml fermentation liquid in a 250ml flat-bottom conical bottle to perform aerobic fermentation, the fourth group uses 50ml fermentation liquid in a 250ml baffle conical bottle to perform aerobic fermentation, and the fifth group uses 30ml fermentation liquid in a 250ml baffle conical bottle to perform aerobic fermentation. The dissolved oxygen of the first group to the fifth group is gradually increased. Five shake flasks were fermented at 37℃for three days and their enzyme activities were measured.
The results showed that the first group had an enzyme activity of 0.12U/ml, the second group had an enzyme activity of 0.20U/ml, the third group had an enzyme activity of 0.42U/ml, the fourth group had an enzyme activity of 1.26U/ml, and the fifth group had an enzyme activity of 1.64U/ml, wherein the fifth group had the highest enzyme activity, which was about 8-fold higher than the basal fermentation medium. The bacterium is more favorable for producing chitinase under the condition of sufficient oxygen.
Influence of carbon Source and carbon Source addition amount
Powder chitin, glucose, fructose, sucrose, starch, lactose, cellulose powder, xylose and the like are selected as carbon sources to replace colloid chitin on a basic fermentation culture medium, the addition amount of the carbon sources is 1 percent, namely 10g/L, and 1 per mill of colloid chitin is added as an inducer, and the fermentation is carried out for 3 days at 37 ℃ by using a baffle conical flask.
The results are shown in FIG. 3. As can be seen from FIG. 3, the effect of the powdered chitin as a carbon source is best, and the enzyme activity of the crude fermentation broth is 1.82U/ml. The chitin powder has low preparation cost and wide source, and is a proper carbon source.
Under the condition that other conditions are not changed, the adding amount of the carbon source (chitin powder) is changed, and fermentation is performed under the same conditions. The results showed that the enzyme activity was highest when 3% chitin powder was added, and at this time the enzyme activity was 2.24U/ml. Therefore, the addition of a carbon source in fermentation culture is advantageous for improving the enzyme activity.
Example 4: preparation and detection of chitosan oligosaccharide
Preparation of crude enzyme solution: firstly, inoculating glycerol bacteria or flat bacteria into a fermentation culture medium for activation for 1 day, inoculating 1% of seed solution into the fermentation culture medium, and culturing at 37 ℃ and 200rpm for three days, wherein the composition of the fermentation culture medium (g/L) is as follows: chitin powder 30, peptone 10, naCl 2.5, KH 2 PO 4 0.7，K 2 HPO 4 0.3，MgSO 4 0.5, pH7.0, and 1% colloidal chitin as inducer. The resulting fermentation broth was centrifuged at 8000rpm for 10min and the supernatant was collected. Taking 10U of crude enzyme solution, adding 100mL of 1% colloidal chitin, and reacting for 24 hours in a constant temperature water bath kettle at 50 ℃ while stirring. After the reaction was completed, the mixture was centrifuged at 10000rpm for 10 minutes to remove some impurities remained and unreacted substrates, and the supernatant was collected and identified.
TLC identification: first, a straight line is drawn 1cm away from the top of the thin layer chromatography plate, and one sample is drawn every 1cm, and one sample is 3. Mu.l of enzymolysis product. And placing the sample in a spreading cylinder for spreading after spotting. The developing agent formula (v/v) is isopropanol: water: ammonia = 15:1:7.5. the color reagent formula (v/v) is anisaldehyde: ethanol: concentrated sulfuric acid: acetic acid = 5:90:5:1.
the identification result is shown in figure 4. The results show that the final products are chitins, disaccharides and trisaccharides.
Example 5: analysis of enzymatic products under different conditions
In this example, two enzyme groups were obtained by shake-flask fermentation with a flat bottom and a baffle plate, and the fermentation medium and fermentation method were the same as those in example 4. The crude enzyme produced by flat bottom shake flask fermentation is numbered 1, the crude enzyme produced by baffle shake flask fermentation is numbered 2, and the fermentation conditions of the two groups of enzymes are identical except for different shake flasks. The enzyme activities and products of the reactions 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 the enzyme activities are shown in Table 1, and thus, the optimal reaction temperatures for the enzyme No.1 and the enzyme No. 2 were 50 ℃.
TABLE 1 enzyme activities at different enzymatic hydrolysis temperatures
And then the substrate is subjected to enzymolysis by the No.1 enzyme and the No. 2 enzyme, and the products and the yields of the substrate at different temperatures are measured. The specific method comprises the following steps:
2ml of 1% colloidal chitin, namely 20mg of substrate, is taken, 200ul of No.1 or No. 2 crude enzyme solution is added, enzymolysis is carried out for 24 hours at 30 ℃,40 ℃,50 ℃ and 60 ℃ respectively, the product composition is detected by TLC, and the content of each component is detected by HPLC.
TLC results showed: enzyme No.1 is mainly produced in the presence of chitobiose under different temperature conditions. Enzyme No. 2 can produce mainly disaccharide at 50 ℃. The sugar content of enzyme No.1 and enzyme No. 2 after enzymolysis at 50℃was quantitatively determined by HPLC, and the results are shown in tables 2 and 3.
Table 21 enzyme hydrolysis at 50℃for 24h
Table 3 2 enzyme hydrolysis at 50℃for 24h
Degradation rate = mass of each sugar after degradation/total mass of substrate
Sugar ratio = sugar content/total sugar content x 100%
Each sugar concentration= (standard concentration x sample peak area)/standard peak area;
wherein the standard substance refers to a pure chitosan oligosaccharide standard substance, and the sample refers to an enzymolysis sample obtained through experiments.
Therefore, the products and the yields are different under different conditions, so that the yield and the proportion of the chitosan oligosaccharide can be controlled by controlling the enzymolysis temperature and the fermentation condition.
FIG. 5 shows TLC patterns of products of enzyme No.1 and enzyme No. 2 at different enzymolysis temperatures.
FIG. 6 is a liquid phase diagram of the No.1 enzyme enzymatic hydrolysis product at 50 ℃.
FIG. 7 is a liquid phase diagram of the No. 2 enzyme enzymatic hydrolysis product at 50 ℃.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments 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-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (3)
1. A strain for producing chitinase is characterized in that the strain is a new strain of genus Chitinilyticum of class Neisseriaceae, named Chitinilyticum sp.ZQ-8, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2021418 and the preservation date of 2021, 4 months and 20 days.
2. Use of the chitinase-producing strain of claim 1 for producing chitinase, wherein the chitinase-producing strain of claim 1 is used for degrading chitin to produce chitooligosaccharides.
3. The use according to claim 2, wherein the crude enzyme enzymatic hydrolysate produced by fermentation in a flat bottom shake flask is mainly chitosan and the crude enzyme enzymatic hydrolysate produced by fermentation in a baffle shake flask is mainly chitosan and chitosan.
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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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| 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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