CN104531639A - Bacteriostatic chitin enzymolysis method - Google Patents
Bacteriostatic chitin enzymolysis method Download PDFInfo
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- CN104531639A CN104531639A CN201410785605.6A CN201410785605A CN104531639A CN 104531639 A CN104531639 A CN 104531639A CN 201410785605 A CN201410785605 A CN 201410785605A CN 104531639 A CN104531639 A CN 104531639A
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- Prior art keywords
- chitin
- chitinase
- enzyme solution
- solution according
- enzymolysis
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- 229920002101 Chitin Polymers 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title abstract description 22
- 230000003385 bacteriostatic effect Effects 0.000 title abstract 3
- 102000004190 Enzymes Human genes 0.000 claims abstract description 59
- 108090000790 Enzymes Proteins 0.000 claims abstract description 59
- 108010022172 Chitinases Proteins 0.000 claims abstract description 23
- 102000012286 Chitinases Human genes 0.000 claims abstract description 23
- 238000002360 preparation method Methods 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
- 239000000843 powder Substances 0.000 claims abstract description 16
- 238000011084 recovery Methods 0.000 claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 10
- 238000003756 stirring Methods 0.000 claims abstract description 10
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 7
- 241000894006 Bacteria Species 0.000 claims abstract description 6
- 238000010438 heat treatment Methods 0.000 claims abstract description 5
- 244000005700 microbiome Species 0.000 claims abstract description 5
- 229940088598 enzyme Drugs 0.000 claims description 55
- 239000007788 liquid Substances 0.000 claims description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 238000010521 absorption reaction Methods 0.000 claims description 14
- 239000003463 adsorbent Substances 0.000 claims description 14
- 230000000844 anti-bacterial effect Effects 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 10
- 239000012190 activator Substances 0.000 claims description 8
- 238000011218 seed culture Methods 0.000 claims description 7
- 239000004382 Amylase Substances 0.000 claims description 6
- 108010065511 Amylases Proteins 0.000 claims description 6
- 102000013142 Amylases Human genes 0.000 claims description 6
- 108010059892 Cellulase Proteins 0.000 claims description 6
- 108010059820 Polygalacturonase Proteins 0.000 claims description 6
- 235000019418 amylase Nutrition 0.000 claims description 6
- 229940106157 cellulase Drugs 0.000 claims description 6
- 238000002425 crystallisation Methods 0.000 claims description 6
- 230000008025 crystallization Effects 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 229940059442 hemicellulase Drugs 0.000 claims description 6
- 108010002430 hemicellulase Proteins 0.000 claims description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 4
- 241000030294 Chitinolyticbacter Species 0.000 claims description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 2
- 239000000047 product Substances 0.000 abstract description 26
- 239000011347 resin Substances 0.000 abstract description 9
- 229920005989 resin Polymers 0.000 abstract description 9
- 230000008569 process Effects 0.000 abstract description 5
- 230000000593 degrading effect Effects 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000004071 biological effect Effects 0.000 abstract description 2
- 238000005265 energy consumption Methods 0.000 abstract description 2
- 239000003480 eluent Substances 0.000 abstract 2
- 230000002255 enzymatic effect Effects 0.000 abstract 2
- 239000000413 hydrolysate Substances 0.000 abstract 2
- 230000003213 activating effect Effects 0.000 abstract 1
- 230000002051 biphasic effect Effects 0.000 abstract 1
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 230000002349 favourable effect Effects 0.000 abstract 1
- 230000000813 microbial effect Effects 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 238000001179 sorption measurement Methods 0.000 abstract 1
- 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 12
- 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 12
- 230000001580 bacterial effect Effects 0.000 description 11
- 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 10
- 239000008103 glucose Substances 0.000 description 10
- 238000011169 microbiological contamination Methods 0.000 description 8
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 239000004160 Ammonium persulphate Substances 0.000 description 5
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 5
- 235000019395 ammonium persulphate Nutrition 0.000 description 5
- 235000015278 beef Nutrition 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000000502 dialysis Methods 0.000 description 5
- 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 5
- 239000012153 distilled water Substances 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- 229940066779 peptones Drugs 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- BLSQLHNBWJLIBQ-OZXSUGGESA-N (2R,4S)-terconazole Chemical compound C1CN(C(C)C)CCN1C(C=C1)=CC=C1OC[C@@H]1O[C@@](CN2N=CN=C2)(C=2C(=CC(Cl)=CC=2)Cl)OC1 BLSQLHNBWJLIBQ-OZXSUGGESA-N 0.000 description 4
- 229940089256 fungistat Drugs 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000004064 recycling Methods 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
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 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
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- 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)
-
- C—CHEMISTRY; METALLURGY
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a bacteriostatic chitin enzymolysis method which comprises the following steps: 1, preparation of chitinase through microbial fermentation: by taking a chitinase preparation microorganism as a test bacterium, fermenting to prepare chitinase, obtaining chitinase crude enzyme after the fermentation is finished, and purifying, dialyzing and concentrating to obtain chitin degrading enzyme; 2, enzymolysis of chitin: putting the chitin degrading enzyme in a reaction kettle, adding chitin powder, a metal ion activating agent, an auxiliary enzyme preparation and an organic reagent, and heating at constant temperature and stirring to obtain enzymatic hydrolysate when the chitin powder is completely degraded; and 3, recovery of product: centrifuging the enzymatic hydrolysate to obtain a biphasic system, wherein the upper system is an organic system and the lower system is a water system, adsorbing by adsorption resins, eluting, collecting the eluent and treating the eluent to obtain the product. According to the bacteriostatic chitin enzymolysis method, the raw materials can be repeatedly utilized and the conversion process is sterile so that high yield can be obtained when the energy consumption is reduced; and the degraded product is favorable in biological activity so that remarkable economic benefit is provided.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of antibacterial chitin enzyme solution.
Background technology
Sugar industry is the basic industry of food service industry, is also the primary industry of the products such as chemical industry, fermentation, medicine.In data display 2012 to 2013 year, China's candy output reaches 1306.8 ten thousand tons, and wherein as the sunrise industry of technical field of food biotechnology, function carbohydrates and their derivative industry develops swift and violent in recent years.Chitin is only second to cellulosic polysaccharide as a kind of nature content, and its degraded product and derivative thereof are widely used in the various fields such as food, medicine, environmental protection, agricultural, daily use chemicals, have defined the huge market requirement.But the production of chitin degrading product and derivative thereof mostly is chemical method at present.Reaction process needs to use strong acid and strong base, and process is difficult to control, and causes great pollution to environment.
Enzymic degradation chitin prepares glucosamine, and to have working condition gentleness controlled, and environment amenable advantage is current study hotspot.But the carbon source that chitin degrading product both can utilize as thalline, can be used as nitrogenous source again.Conversion process is difficult to control, very easily microbiological contamination.Microbiological contamination during the fermentation, can make tunning 2-Acetamido-2-deoxy-D-glucose/chitin oligo saccharide by contaminate miscellaneous bacteria and consume rapidly, increase the separation and Extraction difficulty of product, affect product quality level.
The microbiological contamination measure that prevents conventional in current industrial application mainly utilizes ultraviolet sterilization device, ultrasonic sterilization device, autoclave etc., a kind of preventing of such as Chinese patent utilizes airtight connection ultraviolet sterilization device or ultrasonic sterilization device in reaction system in the novel method of bacterial contamination on materials for producing sugar alcohol (application number: CN200910256591.8), sterilizing is carried out to feed liquid, reaches without dye effect.Chinese patent is a kind of reduces continuously fermenting to spread cultivation in device (CN201220624316.4) and utilizing steam moist heat sterilization to reach fungistatic effect of microbiological contamination probability, and reduce open amount by merging pipeline, and reduce valve quantity, reduce the microbiological contamination probability of the process of spreading cultivation of continuously fermenting.Although above-mentioned Technical comparing is ripe, there is apparatus expensive, the shortcoming of operational hazards, limit the development of biological sugar-refining industry.
Summary of the invention
For the deficiencies in the prior art, the object of the present invention is to provide a kind of antibacterial chitin enzyme solution, simple to operate, little to the side effect of enzymolysis, and product is easily separated.
For solving prior art problem, the technical solution used in the present invention:
An antibacterial chitin enzyme solution, comprises the following steps:
Step 1, fermentable produces chitinase
To produce chitinase microorganism for supplying examination bacterium, to be inoculated on seed culture medium for examination bacterium, at 37 DEG C, 200rpm initial pH cultivates under the condition of 6 ~ 8, obtain seed liquor, again seed liquor is inoculated in the fermentor tank being equipped with and producing enzymic fermentation substratum, at 30 DEG C, 200 rpm, initial pH is 6 ~ 8, the condition bottom fermentation of air flow 1vvm, obtains chitinase crude enzyme liquid, after purifying, dialyse, concentrating, obtain chitin-degrading enzyme;
Step 2, enzymolysis chitin
Be placed in by chitin-degrading enzyme in reactor, add chitin powder, metal ion activator, auxiliary enzymes preparation and organic reagent successively, thermostatically heating also stirs, and treats that chitin powder is degraded completely, obtains enzymolysis solution;
Step 3, the recovery of product
Centrifugal enzymolysis solution, obtains diphasic system, its at the middle and upper levels system be organic system, lower floor is water system, with polymeric adsorbent absorption, wash-out, collect elutriant after cryoconcentration, alcohol wash, crystallization, drying, obtain product.
As preferably, step 2, described metal ion activator is Mg
2+, Mn
2+, Ca
2+, Na
+, K
+, Al
3+, Fe
2+, Fe
3+, Zn
2+or Cu
2+one or more in metal-salt.
As preferably, step 2, the final concentration of described metal ion activator is 5-25 mmol/L.
As preferably, step 2, described auxiliary enzymes preparation comprises cellulase 2-60%, hemicellulase 1-15%, amylase 5-70%, polygalacturonase 2-40% by mass percentage.
As preferably, step 2, described auxiliary enzymes preparation accounts for the 0.1-0.3% of reaction system total mass.
As preferably, described organic reagent is one or more in acetone, ether, normal hexane, hexanaphthene, methylene dichloride or sherwood oil.
As preferably, step 2, the temperature of thermostatically heating is 20-37 DEG C, and stirring velocity is 50-200 rpm.
As preferably, the volume fraction that described organic reagent accounts for total system is 10-70%.
As preferably, described microorganism is
chitinolyticbacter meiyuanensissYBC-H1 or
chitinibactersp.GC72.
beneficial effect
(1) by adding chitinase activator and auxiliary enzymes preparation in chitinase enzymatic hydrolysis system, enzymolysis efficiency can be made to improve about 30%.
(2) employing adds organic reagent in transformation system, effectively can prevent microbiological contamination, reduce potential safety hazard.Improve enzymolysis speed, accelerate reaction process, shortened for 12 h reaction times.
(3) organic reagent is after enzymolysis terminates, and through simple centrifugally operated and recovery, can realize recycling, avoid the pollution to environment, reduce production cost, have promotional value.
Embodiment
The following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.
embodiment 1
Step 1, fermentable produces chitinase
With
chitinolyticbacter meiyuanensissYBC-H1 bacterial strain is that chitinase produces bacterial strain, and seed culture medium is (2 g/L glucose, 2 g/L peptones, 0.7 g/L KH
2pO
4, 0.3 g/L K
2hPO
43H
2o, 0.4 g/L MgSO
47H
2o, pH 7.0), 37 DEG C, 200 rpm cultivate 12 h.By seed liquor with the inoculum size of volume fraction 8%, access 7.5 L and the fermentor tank producing enzymic fermentation substratum is housed, liquid amount 4 L, culture medium is (3 g/L powder chitins, 3 g/L glucose, 3 g/L beef extracts, 0.7 g/L KH
2pO
4, 0.3 g/L K
2hPO
43H
2o, 0.5 g/L MgSO
47H
2o) culture temperature 30 DEG C, rotating speed is 200 rpm, air flow 1 vvm, terminates fermentation after initial pH 7.5,72 h.
By centrifugal for fermented liquid 8000g, abandon thalline, precipitate through ammonium persulphate after collecting supernatant liquor, redissolve.Loaded in dialysis tubing (molecular weight cut-off 8000 KDa) by chitin crude enzyme liquid, take distilled water as dialyzate, change a dialyzate every 6 h, after 24 h that dialyse under 4 DEG C of conditions, concentrated enzyme liquid, obtains chitin-degrading enzyme after concentrated.
Step 2, enzymolysis chitin
In 1 L reactor, add chitin-degrading enzyme 90 U, final concentration is the chitin powder of 30 g/L, and adds CaCl successively
2solution 10mM, auxiliary enzymes preparation accounts for 0.1% of reaction system total mass, and wherein, auxiliary enzymes preparation is by the following material by massfraction: cellulase 30%, hemicellulase 10%, amylase 2 0%, polygalacturonase 40% form.Add again account for total system volume fraction 40% normal hexane as fungistat, under 37 DEG C of constant temperatures, 200 rpm stir conversion 84 h, obtain enzymolysis solution.
Step 3, the recovery of product
After enzymolysis solution 3000g centrifugal treating, upper strata organic system normal hexane reclaims, and the rate of recovery can reach 95%.After removing bottom enzymolysis residue, lower floor's water system adopts the required 2-Acetamido-2-deoxy-D-glucose of method absorption of polymeric adsorbent absorption, after cryoconcentration, alcohol wash, crystallization, drying, obtain product.Wherein, resin absorption can adopt strong Polar Adsorbent Resin, low-pole polymeric adsorbent, flow velocity 30-450 mL/h, on-line ultraviolet detector 210 nm wavelength detecting effluent liquid, adopts the method wash-out adsorbed product of graded ethanol wash-out.The 2-Acetamido-2-deoxy-D-glucose purity obtained through this method of Liquid Detection is high, and after 84 h transform, yield can reach 95%.
embodiment 2
Step 1, fermentable produces chitinase
With
chitinibactersp.GC72 bacterial strain is that chitinase produces bacterial strain, and seed culture medium is (4 g/L glucose, 4 g/L peptones, 0.7 g/L KH
2pO
4, 0.3 g/L K
2hPO
43H
2o, 0.4 g/L MgSO
47H
2o, pH 7.0), 37 DEG C, 200 rpm cultivate 12 h, seed liquor is equipped with the 7.5 L fermentor tanks producing enzymic fermentation substratum, liquid amount 4 L with the access of volume fraction 6% inoculum size, and culture medium is (4 g/L powder chitins, 2 g/L glucose, 4 g/L beef extracts, 0.7 g/L KH
2pO
4, 0.3 g/L K
2hPO
43H
2o, 0.5 g/L MgSO
47H
2o) culture temperature 30 DEG C, rotating speed is 200 rpm, air flow 1 vvm, and initial pH 7.5,72 h terminates fermentation and collects fermented liquid.
By centrifugal for fermented liquid 8000g, abandon thalline, precipitate through ammonium persulphate after collecting supernatant liquor, redissolve.Loaded in dialysis tubing (molecular weight cut-off 8000 KDa) by chitin crude enzyme liquid, take distilled water as dialyzate, change a dialyzate every 6 h, after 24 h that dialyse under 4 DEG C of conditions, concentrated enzyme liquid, obtains chitin-degrading enzyme after concentrated.
Step 2, enzymolysis chitin
In 1 L reactor, add chitin-degrading enzyme 90 U, final concentration is the chitin powder of 30 g/L, in reactor, add MgSO
4solution 15mM, auxiliary enzymes preparation accounts for 0.2% of reaction system total mass, and wherein, auxiliary enzymes preparation is by the following material by massfraction: cellulase 30%, hemicellulase 10%, and amylase 2 0%, polygalacturonase 40% forms.Add again account for total system volume fraction 60% normal hexane as fungistat, under 37 DEG C of constant temperatures, 200 rpm stir conversion 84 h, obtain enzymolysis solution.
Step 3, the recovery of product
After enzymolysis solution 3000g centrifugal treating, upper strata organic system normal hexane reclaims, and the rate of recovery can reach 95%.After removing bottom enzymolysis residue, lower floor's water system adopts the required 2-Acetamido-2-deoxy-D-glucose of method absorption of polymeric adsorbent absorption, after cryoconcentration, alcohol wash, crystallization, drying, obtain product.Wherein, resin absorption can adopt strong Polar Adsorbent Resin, low-pole polymeric adsorbent, flow velocity 30-450 mL/h, on-line ultraviolet detector 210 nm wavelength detecting effluent liquid, adopts the method wash-out adsorbed product of graded ethanol wash-out.The 2-Acetamido-2-deoxy-D-glucose purity obtained through this method of Liquid Detection is high, and after 24 h transform, yield can reach 40%, and after 84 h transform, yield can reach 90%.
embodiment 3
Step 1, fermentable produces chitinase
With
chitinibactersp.GC72 bacterial strain is that chitinase produces bacterial strain, and seed culture medium is (4 g/L glucose, 4 g/L peptones, 0.7 g/L KH
2pO
4, 0.3 g/L K
2hPO
43H
2o, 0.4 g/L MgSO
47H
2o, pH 7.0), 37 DEG C, 200 rpm cultivate 12 h, seed liquor is equipped with the 7.5 L fermentor tanks producing enzymic fermentation substratum, liquid amount 4 L with the access of volume fraction 6% inoculum size, and culture medium is (4 g/L powder chitins, 2 g/L glucose, 4 g/L beef extracts, 0.7 g/L KH
2pO
4, 0.3 g/L K
2hPO
43H
2o, 0.5 g/L MgSO
47H
2o) culture temperature 30 DEG C, rotating speed is 200 rpm, air flow 1 vvm, and initial pH 7.5,72 h terminates fermentation and collects fermented liquid.
By centrifugal for fermented liquid 8000g, abandon thalline, precipitate through ammonium persulphate after collecting supernatant liquor, redissolve.Loaded in dialysis tubing (molecular weight cut-off 8000 KDa) by chitin crude enzyme liquid, take distilled water as dialyzate, change a dialyzate every 6 h, after 24 h that dialyse under 4 DEG C of conditions, concentrated enzyme liquid, obtains chitin-degrading enzyme after concentrated.
Step 2, enzymolysis chitin
In 1 L reactor, add chitin-degrading enzyme 90 U, final concentration is the chitin powder of 30 g/L, adds and add MgSO in reactor
4solution 15mM, auxiliary enzymes preparation accounts for 0.3% of reaction system total mass, and wherein, auxiliary enzymes preparation is by the following material by massfraction: cellulase 10%, hemicellulase 40%, amylase 2 5%, polygalacturonase 25%.Add again account for total system volume fraction 60% normal hexane as fungistat, under 37 DEG C of constant temperatures, 200 rpm stir conversion 84 h, obtain enzymolysis solution.
Step 3, the recovery of product
After enzymolysis solution is carried out 3000g centrifugal treating, upper strata organic system normal hexane reclaims, and the rate of recovery can reach 95%.After removing bottom enzymolysis residue, lower floor's water system adopts the required 2-Acetamido-2-deoxy-D-glucose of method absorption of polymeric adsorbent absorption, after cryoconcentration, alcohol wash, crystallization, drying, obtain product.Wherein, resin absorption can adopt strong Polar Adsorbent Resin, low-pole polymeric adsorbent, flow velocity 30-450 mL/h, on-line ultraviolet detector 210 nm wavelength detecting effluent liquid, adopts the method wash-out adsorbed product of graded ethanol wash-out.The 2-Acetamido-2-deoxy-D-glucose purity obtained through this method of Liquid Detection is high, and after 24 h transform, yield can reach 48%, and after 96 h transform, yield can reach 99%.
comparative example 1
Step 1, fermentable produces chitinase
With
chitinibactersp.GC72 bacterial strain is that chitinase produces bacterial strain, and seed culture medium is (4 g/L glucose, 4 g/L peptones, 0.7 g/L KH
2pO
4, 0.3 g/L K
2hPO
43H
2o, 0.4 g/L MgSO
47H
2o, pH 7.0), 37 DEG C, 200 rpm cultivate 12 h, seed liquor is equipped with the 7.5 L fermentor tanks producing enzymic fermentation substratum, liquid amount 4 L with the access of volume fraction 6% inoculum size, and culture medium is (4 g/L powder chitins, 2 g/L glucose, 4 g/L beef extracts, 0.7 g/L KH
2pO
4, 0.3 g/L K
2hPO
43H
2o, 0.5 g/L MgSO
47H
2o) culture temperature 30 DEG C, rotating speed is 200 rpm, air flow 1 vvm, and initial pH 7.5,72 h terminates fermentation and collects fermented liquid.
By centrifugal for fermented liquid 8000g, abandon thalline, precipitate through ammonium persulphate after collecting supernatant liquor, redissolve.Loaded in dialysis tubing (molecular weight cut-off 8000 KDa) by chitin crude enzyme liquid, take distilled water as dialyzate, change a dialyzate every 6 h, after 24 h that dialyse under 4 DEG C of conditions, concentrated enzyme liquid, obtains chitin-degrading enzyme after concentrated.
Step 2, enzymolysis chitin
In 1 L reactor, add chitin-degrading enzyme 90 U, final concentration is the chitin powder of 30 g/L, and under 37 DEG C of constant temperatures, 200 rpm stir conversion 84 h.
Step 3, the recovery of product
After enzymolysis solution 3000g centrifugal treating, through the 2-Acetamido-2-deoxy-D-glucose that Liquid Detection obtains, after transforming during 24 h, yield can reach 35%, and due to system microbiological contamination, 96 h do not detect the existence of 2-Acetamido-2-deoxy-D-glucose.
comparative example 2
Step 1, fermentable produces chitinase
With
chitinibactersp.GC72 bacterial strain is that chitinase produces bacterial strain, and seed culture medium is (4 g/L glucose, 4 g/L peptones, 0.7 g/L KH
2pO
4, 0.3 g/L K
2hPO
43H
2o, 0.4 g/L MgSO
47H
2o, pH 7.0), 37 DEG C, 200 rpm cultivate 12 h, seed liquor is equipped with the 7.5 L fermentor tanks producing enzymic fermentation substratum, liquid amount 4 L with the access of volume fraction 6% inoculum size, and culture medium is (4 g/L powder chitins, 2 g/L glucose, 4 g/L beef extracts, 0.7 g/L KH
2pO
4, 0.3 g/L K
2hPO
43H2O, 0.5 g/L MgSO
47H
2o) culture temperature 30 DEG C, rotating speed is 200 rpm, air flow 1 vvm, and initial pH 7.5,72 h terminates fermentation and collects fermented liquid.
By centrifugal for fermented liquid 8000g, abandon thalline, precipitate through ammonium persulphate after collecting supernatant liquor, redissolve.Loaded in dialysis tubing (molecular weight cut-off 8000 KDa) by chitin crude enzyme liquid, take distilled water as dialyzate, change a dialyzate every 6 h, after 24 h that dialyse under 4 DEG C of conditions, concentrated enzyme liquid, obtains chitin-degrading enzyme after concentrated.
Step 2, enzymolysis chitin
In 1 L reactor, add chitin-degrading enzyme 90 U, final concentration is the chitin powder of 30 g/L, adds and add MgSO in reactor
4solution 15mM, auxiliary enzymes preparation accounts for 0.3% of reaction system total mass, and wherein, auxiliary enzymes preparation is by the following material by massfraction: cellulase 50%, hemicellulase 10%, amylase 10%, and polygalacturonase 30% forms.The normal hexane adding volume fraction 60% is again as fungistat, and under 20 DEG C of conditions, 200 rpm stir conversion 84 h, obtain enzymolysis solution.
Step 3, the recovery of product
After enzymolysis solution 3000g centrifugal treating, upper strata organic system normal hexane reclaims, and the rate of recovery can reach 95%.After removing bottom enzymolysis residue, lower floor's water system adopts the required 2-Acetamido-2-deoxy-D-glucose of method absorption of polymeric adsorbent absorption, after cryoconcentration, alcohol wash, crystallization, drying, obtain product.Wherein, resin absorption can adopt strong Polar Adsorbent Resin, low-pole polymeric adsorbent, flow velocity 30-450 mL/h, on-line ultraviolet detector 210 nm wavelength detecting effluent liquid, adopts the method wash-out adsorbed product of graded ethanol wash-out.The 2-Acetamido-2-deoxy-D-glucose purity obtained through this method of Liquid Detection is high, and after 24 h transform, yield is that after 28%, 96 h transform, yield is 71%.
Known by the result of above-described embodiment and comparative example, antibacterial material of the present invention can realize recycling, and conversion process is aseptic, high yield is obtained while reducing energy consumption, wherein, the interpolation of chitinase activator and auxiliary enzymes preparation, can make enzymolysis efficiency improve about 30%.The application of organic reagent can suppress transformation system microbiological contamination, make the yield of the transformation system 2-Acetamido-2-deoxy-D-glucose after 96 h by 0 most promotion to 99%, and degraded product biological activity is good, has significant economic benefit.
Claims (9)
1. an antibacterial chitin enzyme solution, is characterized in that, comprise the following steps:
Step 1, fermentable produces chitinase
To produce chitinase microorganism for supplying examination bacterium, will be inoculated on seed culture medium, at 37 DEG C for examination bacterium, 200 rpm, initial pH cultivates under the condition of 6 ~ 8, obtains seed liquor, then seed liquor is inoculated in the fermentor tank that is equipped with and produces enzymic fermentation substratum, at 30 DEG C, 200 rpm, initial pH are 6 ~ 8, the condition bottom fermentation of air flow 1vvm, obtain chitinase crude enzyme liquid, after purifying, dialyse, concentrating, obtain chitin-degrading enzyme;
Step 2, enzymolysis chitin
Be placed in by chitin-degrading enzyme in reactor, add chitin powder, metal ion activator, auxiliary enzymes preparation and organic reagent successively, thermostatically heating also stirs, and treats that chitin powder is degraded completely, obtains enzymolysis solution;
Step 3, the recovery of product
Centrifugal enzymolysis solution, obtains diphasic system, its at the middle and upper levels system be organic system, lower floor is water system, lower floor's water system with polymeric adsorbent absorption, wash-out, collect elutriant after cryoconcentration, alcohol wash, crystallization, drying, obtain product.
2. antibacterial chitin enzyme solution according to claim 1, is characterized in that: step 2, and described metal ion activator is Mg
2+, Mn
2+, Ca
2+, Na
+, K
+, Al
3+, Fe
2+, Fe
3+, Zn
2+or Cu
2+one or more in metal-salt.
3. antibacterial chitin enzyme solution according to claim 1, is characterized in that: step 2, and the final concentration of described metal ion activator is 5-25 mmol/L.
4. antibacterial chitin enzyme solution according to claim 1, is characterized in that: step 2, and described auxiliary enzymes preparation comprises following cellulase 2-60%, hemicellulase 1-15%, amylase 5-70%, polygalacturonase 2-40% by mass percentage.
5. antibacterial chitin enzyme solution according to claim 1, is characterized in that: step 2, and described auxiliary enzymes preparation accounts for the 0.1-0.3% of reaction system total mass.
6. antibacterial chitin enzyme solution according to claim 1, is characterized in that: described organic reagent is one or more in acetone, ether, normal hexane, hexanaphthene, methylene dichloride or sherwood oil.
7. antibacterial chitin enzyme solution according to claim 1, is characterized in that: step 2, and the temperature of thermostatically heating is 20-37 DEG C, and stirring velocity is 50-200 rpm.
8. antibacterial chitin enzyme solution according to claim 1, is characterized in that: the volume fraction that described organic reagent accounts for total system is 10-70%.
9. antibacterial chitin enzyme solution according to claim 1, is characterized in that: described microorganism is
chitinolyticbacter meiyuanensissYBC-H1 or
chitinibactersp.GC72.
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| CN108559765A (en) * | 2017-12-28 | 2018-09-21 | 南京工业大学 | A kind of method that biological enzyme extracts N-acetylglucosamine and astaxanthin from cray shell |
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