CN104130961A - Bacterial strain for producing chitinase and its use in chitin enzymolysis - Google Patents
Bacterial strain for producing chitinase and its use in chitin enzymolysis Download PDFInfo
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- CN104130961A CN104130961A CN201410361719.8A CN201410361719A CN104130961A CN 104130961 A CN104130961 A CN 104130961A CN 201410361719 A CN201410361719 A CN 201410361719A CN 104130961 A CN104130961 A CN 104130961A
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- 229920002101 Chitin Polymers 0.000 title claims abstract description 39
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 37
- 102000012286 Chitinases Human genes 0.000 title claims abstract description 26
- 108010022172 Chitinases Proteins 0.000 title claims abstract description 26
- 102000004190 Enzymes Human genes 0.000 claims abstract description 54
- 108090000790 Enzymes Proteins 0.000 claims abstract description 54
- 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 claims abstract description 15
- 238000000855 fermentation Methods 0.000 claims abstract description 12
- 230000004151 fermentation Effects 0.000 claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 28
- 239000000843 powder Substances 0.000 claims description 15
- 229910019142 PO4 Inorganic materials 0.000 claims description 14
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 14
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 14
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 14
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 14
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 14
- 239000010452 phosphate Substances 0.000 claims description 14
- 229910052700 potassium Inorganic materials 0.000 claims description 14
- 239000011591 potassium Substances 0.000 claims description 14
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 11
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims 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 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 230000007062 hydrolysis Effects 0.000 claims description 7
- 238000006460 hydrolysis reaction Methods 0.000 claims description 7
- 240000008892 Helianthus tuberosus Species 0.000 claims description 6
- 235000003230 Helianthus tuberosus Nutrition 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 239000011777 magnesium Substances 0.000 claims description 4
- 239000012092 media component Substances 0.000 claims description 2
- 235000015097 nutrients Nutrition 0.000 claims description 2
- 239000000047 product Substances 0.000 abstract description 21
- 238000004321 preservation Methods 0.000 abstract description 5
- 238000004458 analytical method Methods 0.000 abstract description 2
- 241000843811 Chitinibacter sp. GC72 Species 0.000 abstract 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 abstract 1
- 230000002255 enzymatic effect Effects 0.000 abstract 1
- 239000000413 hydrolysate Substances 0.000 abstract 1
- 229950006780 n-acetylglucosamine Drugs 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 24
- 241000894006 Bacteria Species 0.000 description 17
- 238000000034 method Methods 0.000 description 17
- 239000001963 growth medium Substances 0.000 description 15
- 238000012216 screening Methods 0.000 description 15
- 230000012010 growth Effects 0.000 description 8
- 241000208125 Nicotiana Species 0.000 description 7
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 244000052769 pathogen Species 0.000 description 7
- 230000001717 pathogenic effect Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 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 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 241001052560 Thallis Species 0.000 description 4
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 4
- 230000000593 degrading effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 229960002442 glucosamine Drugs 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 239000012533 medium component Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 238000009631 Broth culture Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000238424 Crustacea Species 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- 241000237502 Ostreidae Species 0.000 description 2
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 2
- 230000009603 aerobic growth Effects 0.000 description 2
- MBLBDJOUHNCFQT-LXGUWJNJSA-N aldehydo-N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 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 2
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- 239000003085 diluting agent Substances 0.000 description 2
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- 238000001802 infusion Methods 0.000 description 2
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- 235000013372 meat Nutrition 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Polymers 0.000 description 2
- 125000001477 organic nitrogen group Chemical group 0.000 description 2
- 235000020636 oyster Nutrition 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
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- 150000003839 salts Chemical class 0.000 description 2
- 238000012807 shake-flask culturing Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
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- 235000014347 soups Nutrition 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 241000607516 Aeromonas caviae Species 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 241000192128 Gammaproteobacteria Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000588656 Neisseriaceae Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 241001314279 Zoopagales Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a bacterial strain for producing chitinase. Through classification identification, the bacterial strain is named as Chitinibacter sp.GC72, is preserved in the China center for type culture collection (CCTCC) on April 1, 2014 and has a preservation number of CCTCC NO: M2014113. The invention discloses culture conditions and fermentation enzyme-production conditions suitable for the bacterial strain. Enzymatic property and hydrolysate analysis on the crude enzyme proves that the chitinase secreted by the bacterial strain can effectively degrade chitin and the degraded products mainly comprise N-acetyl-D-glucosamine.
Description
Technical field
The invention belongs to biological technical field, particularly a strain screens from nature the efficient chitin degrading bacterium strain and the application of this bacterial strain enzymolysis chitin that obtain.
Background technology
Chitin, another name chitin, is mainly present in Crustaceans shell, insect shell, fungal cell wall, is that occurring in nature content is only second to cellulosic second largest natural high moleculer eompound, be formed by connecting by β-Isosorbide-5-Nitrae-glycosidic link by monomer 2-acetylamino-2-deoxy-D-glucose.Chitin can generate multi-products after degraded, mainly comprises chitin oligo saccharide, 2-acetylamino-2-deoxy-D-glucose, chitosan, glucosamine etc.These products are widely used in fields such as chemical industry, food, medicine, material, agriculturals at present.By 2011, exceed 2,000,000,000 dollars as the glucosamine of nutritious supplementary and the global marketing volume of derivative product thereof, expect 2017, global glucosamine output can reach 46,600 tons.
Current 85% commercial glucosamine product is to obtain by the shell of acid hydrolysis ocean crustacean.Acid system degrade chitin can produce a large amount of acid-base waste fluids, not only can be to environment, and also degradation process is difficult to control, and product mixes, and biologic activity is difficult to ensure.Also have at present and attempt utilizing microwave irradiation hydrolysis chitin, can obtain the product of different molecular weight by controlled hydrolysis time and microwave intensity, but not only manufacturing requirements is high for this method, is difficult to promote, and may causes actual bodily harm to operator.The focus of research is enzymic degradation chitin at present, and it has mild condition, and cost is low, its lytic activity is high, eco-friendly advantage, has huge potentiality aspect chitin higher value application, be one of study hotspot of current functional foodstuff, Materials science and medicine and other fields.For example, Chinese patent CN 03112043.1 discloses a kind of method of utilizing Aeromonas caviae enzymolysis chitin to obtain chitin oligosaccharide.The main products of biological enzyme degrade chitin is to produce chitinous oligomers at present, obtains the 2-acetylamino-2-deoxy-D-glucose that purity is higher still more difficult.
Summary of the invention
The first object of the present invention is to provide the chitinous bacterial strain of a high-efficiency degradation, utilizes the secreted chitinase degradable chitin of this bacterium to obtain 2-acetylamino-2-deoxy-D-glucose.
For achieving the above object, the technical solution used in the present invention is as follows:
The bacterial strain of chitinase is produced in one strain, it is characterized in that, its Classification And Nomenclature is
chitinibactersp. GC72, has been preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO:M 2014113 on April 1st, 2014.
Morphology and the physiological and biochemical property of bacterial strain of the present invention are as follows:
Colony colour: oyster white.
Aerobic mode: aerobic growth.
Bacterium colony size: 1.9 × 0.8 μ m
Suitable growth temperature: 34-40 DEG C.
Suitable growth pH:6-8
Thalli morphology: rod-short, camber are curved.
Gramstaining: feminine gender.
Salt tolerant growth scope: 0%-1% NaCl.
Above-mentioned
chitinibactersp. GC72 screening method, specifically comprises the steps:
(1) sample collecting
We from Fuyang, Anhui, two places, Nanjing city obtain soil, rot shrimp shell totally 27 duplicate samples, be stored in time 4 DEG C of preservations for subsequent use.
(2) enrichment culture
Get 50 mL broth cultures and be loaded in 500 mL triangular flasks, access sample 0.1 g to be sieved after sterilizing, shake-flask culture 24 h under 37 DEG C, 200 r/min conditions, obtain the bacterium liquid of enrichment.
(3) dull and stereotyped transparent circle screening
Enrichment culture liquid is filtered with aseptic individual layer filter paper, gradient dilution is to proper concn, draw approximately 0.2 mL diluent and coat screening culture medium flat board, cultivate 5 d for 37 DEG C, bacterial strain point sample multiple sieve on screening culture medium flat board that observe, picking product transparent circle bacterial strain (seeing accompanying drawing 1) will screen acquisition, cultivate 5 d for 37 DEG C, taking thalli growth speed and transparent circle size as standard, preferentially bacterial strain does slant preservation.
(4) shaking flask is sieved again
50 mL shaking flask screening culture medium are loaded in 500 mL triangular flasks, every bottle graft enters 1 ring and sieves again bacterial strain slant strains, 37 DEG C, 200 r/min shaking culture 3 d, the centrifugal crude enzyme liquid that obtains of fermented liquid, measure enzyme activity, final reservation, is produced the highest bacterial strain of enzyme activity, and wherein a strain bacterium chitinase work reaches 0.57 U/mL, called after
chitinibactersp. GC72.
Wherein each medium component is as follows:
Enrichment medium: brain heart infusion meat soup (BHI);
Screening culture medium: tobacco brown spot pathogen, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L that 20% concentration is 0.5%;
Fermention medium: glucose 1 g/L, powder chitin 2 g/L, peptone 1 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L.
Second object of the present invention is to provide above-mentioned bacterial strains and produces the application in chitinase in fermentation.
Preferably, the culture condition of described bacterial strain is glucose 1～4 g/L, peptone 2～10 g/L, yeast extract paste 2～10 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L.Culture temperature is 20～37 DEG C, 200 rpm.
Preferably, the condition of described strain fermentation product enzyme is that nutrient media components is jerusalem artichoke powder 1～8 g/L, peptone 2～8 g/L, extractum carnis 2～10 g/L, powder chitin 1～10 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L.Initial pH:6～8,30 DEG C of leavening temperatures.Rotating speed: 200 rpm, air flow: 0.5～1 vvm.
The 3rd object of the present invention is to provide the application that utilizes bacterial strain of the present invention to produce chitinase enzymolysis chitin production 2-acetylamino-2-deoxy-D-glucose.
Preferably, described enzymatic hydrolysis condition is 20～40 DEG C of hydrolysis temperatures, and further, hydrolysis temperature is 40 DEG C.
Preferably, described enzymatic hydrolysis condition is suitable enzymolysis pH value 6.0～8.0, and further enzymolysis pH value is 6.8-7.2.
Preferably, described enzymatic hydrolysis condition for to add Ca in enzymatic hydrolysis system
2+, Mg
2+, Fe
2+, further for adding Ca
2+.
Preferably, described Ca
2+concentration is 10 mmol/L.
Purification fermention medium enzyme liquid on the basis that culture condition to this bacterium and condition of enzyme production are studied, utilizes the crude enzyme liquid of preparation to carry out chitin hydrolysising experiment.Using the chitin of dissimilar, concentration as substrate, by liquid phase, mass spectroscopy enzymolysis product.
Beneficial effect of the present invention:
The secreted chitinase degradable chitin of bacterial strain bacterium of the present invention obtains 2-acetylamino-2-deoxy-D-glucose, through the optimization of fermention medium component and culture condition, under 1 L fermentation system, the enzyme work of this bacterium crude enzyme liquid is up to 0.9 U/mL, the chitinase that produces degrade chitin rapidly, wherein, the product yield of 0.5% concentration tobacco brown spot pathogen 2 h of degrading reaches 86.16%, the product yield of degraded powder chitin (particle diameter 150 nm) 12 h reaches 65.36%, and degraded product purity is high, enzymolysis product main component through liquid phase and mass spectroscopy 0.5% tobacco brown spot pathogen is 2-acetylamino-2-deoxy-D-glucose, without chitobiose.
Brief description of the drawings
Fig. 1 is
chitinibactersp. the mono-bacterium colony of GC72 produces transparent circle situation in screening culture medium.
Fig. 2 is the suitable enzymolysis pH figure of the crude enzyme liquid after purifying.
Fig. 3 is the liquid phase analysis figure of crude enzyme liquid degrade chitin product.
Fig. 4 is the mass spectroscopy figure of crude enzyme liquid degrade chitin product.
Bacterial strain of the present invention, its Classification And Nomenclature is
chitinibactersp. GC72, has been preserved in Chinese Typical Representative culture collection center (CCTCC), address, China on April 1st, 2014. Wuhan. and Wuhan University, deposit number: CCTCC NO:M 2014113.
Embodiment
Enumerate embodiment below the present invention is further described, but therefore do not limit content of the present invention.
Embodiment is the evaluation index to different carbon nitrogen sources hobby property using dry cell weight (DCW) in substratum as thalline, characterizes thalline and produces the suitable carbon nitrogen source of enzyme with enzyme (U/mL) height of living.Result of study show the suitableeest this bacteria growing carbon source, organic nitrogen source, inorganic nitrogen-sourced be respectively glucose, yeast extract, ammonium nitrate; The suitableeest this bacterium produce enzyme carbon source, organic nitrogen source, inorganic nitrogen-sourced be respectively jerusalem artichoke powder, extractum carnis and urea.Live just for this bacterium of index expression is produced chitinase peak enzymolysis-ability temperature, enzymatic hydrolysis system pH, storage stability and the impact of metal ion on enzyme with enzyme in addition.
embodiment 1: the present embodiment explanation
chitinibactersp. the screening method of GC72
chitinibactersp. GC72 screening method, specifically comprises the steps:
(1) sample collecting
We from Fuyang, Anhui, two places, Nanjing city obtain soil, rot shrimp shell totally 27 duplicate samples, be stored in time 4 DEG C of preservations for subsequent use.
(2) enrichment culture
Get 50 mL broth cultures and be loaded in 500 mL triangular flasks, access sample to be sieved after sterilizing, shake-flask culture 24 h under 37 DEG C, 200 r/min conditions, obtain the bacterium liquid of enrichment.
(3) dull and stereotyped transparent circle screening
With aseptic individual layer filter paper, by the filtration of enrichment culture liquid, gradient dilution, to proper concn, is drawn approximately 0.2 mL diluent and is coated screening culture medium flat board, cultivates 5 d for 37 DEG C, and observation, picking produce transparent circle bacterial strain (seeing accompanying drawing 1).The bacterial strain point sample multiple sieve on screening culture medium flat board will screening obtaining, cultivates 5 d for 37 DEG C, and taking thalli growth speed and transparent circle size as standard, preferentially bacterial strain does slant preservation.
(4) shaking flask is sieved again
50 mL shaking flask screening culture medium are loaded in 500 mL triangular flasks, every bottle graft enters 1 ring and sieves again bacterial strain slant strains, 37 DEG C, 200 r/min shaking culture 3 d, the centrifugal crude enzyme liquid that obtains of fermented liquid, measure enzyme activity, final reservation, is produced the highest bacterial strain of enzyme activity, and wherein a strain bacterium chitinase work reaches 0.57 U/mL, called after
chitinibactersp. GC72.
Wherein each medium component is as follows:
Enrichment medium: brain heart infusion meat soup (BHI);
Screening culture medium: tobacco brown spot pathogen, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L that 20% concentration is 0.5%;
Fermention medium: glucose 1 g/L, powder chitin 2 g/L, peptone 1 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L.
embodiment 2: the present embodiment explanation
chitinibactersp. GC72 authentication method
The qualification of this bacterial strain: extract its 16S rDNA of this bacterium postgenome design primer pair and increase and check order, the sequence in sequencing result and GeneBank is compared, find that the pattern formula bacterial strain that this bacterium and its homology are the highest all belongs to
chitinibacterpseudomonas.Judge this bacterium for distortion Gammaproteobacteria, Neisser Zoopagales, Neisseriaceae,
chitinibacterbelong to called after
chitinibactersp.GC72.Form, the physiological and biochemical property of this bacterial strain are as follows:
Colonial morphology and color: circle, oyster white.Thalli morphology: rod-short bending.Gramstaining: feminine gender.Bacterium colony size: 1.9 × 0.8 μ m.Aerobic mode: aerobic growth.Growth temperature: 35-40 DEG C.Growth pH:6-10.Salt tolerant growth scope: 0%-1% NaCl.
embodiment 3: the present embodiment explanation
chitinibactersp. GC72 produces the application in chitinase in fermentation
?seed culture medium: glucose 4 g/L, peptone 4 g/L, yeast extract paste 4 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L.
Culture medium: jerusalem artichoke powder 2 g/L, peptone 4 g/L, extractum carnis 4 g/L, powder chitin 3 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L.
Condition of enzyme production: under 1L fermentor tank system, inoculum size 5%, initial pH:7.5, temperature: 30 DEG C, fermentation period: 96 h, rotating speed: 200 rpm, air flow: 1 vvm.
Under above implementation condition, ferment, and carry out enzyme activity determination: get fermented liquid supernatant enzyme liquid 100 uL, 1% tobacco brown spot pathogen 500 uL, phosphoric acid buffer 1.4 mL of pH 7.0, by above-mentioned system application of sample and mix, add magnetic agitation rotor to be placed under 37 DEG C of water bath with thermostatic control conditions and stir 30 min.Boiling water bath 5 min at once after enzymolysis finishes add DNS reagent 2 mL after system is cooling, mix rear boiling water bath 5 min.Be cooled to centrifugal 5 min of 5000 rpm after room temperature, get supernatant liquor and detect absorbances at 535 nm places, repeat to test three times and obtain mean value, and taking 2-acetylamino-2-deoxy-D-glucose typical curve as according to calculating reducing sugar content.Simultaneously do control experiment with the fermented liquid supernatant enzyme liquid of high-temperature sterilization, enzyme is lived and is defined as under 37 DEG C of conditions per minute and discharges that to be equivalent to the required enzyme amount of 1 μ mol be a Ge Meihuo unit (U).
The crude enzyme liquid enzyme work of gained can reach 0.9 U/mL after measured.
embodiment 4: the present embodiment explanation
chitinibactersp. GC72 produces the application in chitinase in fermentation
Seed culture medium: glucose 1 g/L, peptone 2 g/L, yeast extract paste 10 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L.
Culture medium: jerusalem artichoke powder 8 g/L, peptone 2 g/L, extractum carnis 2 g/L, powder chitin 1 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L.
Condition of enzyme production: under 1L fermentor tank system, inoculum size 5%, initial pH:8, temperature: 37 DEG C, fermentation period: 96 h, rotating speed: 200 rpm, air flow: 1 vvm.
Under above implementation condition, ferment, and carry out enzyme activity determination, method therefor is with embodiment 3.
The crude enzyme liquid enzyme work of gained can reach 0.66 U/mL after measured.
embodiment 5: the present embodiment explanation
chitinibactersp. GC72 produces the application in chitinase in fermentation
Seed culture medium: glucose 4 g/L, peptone 10 g/L, yeast extract paste 2 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L.
Culture medium: jerusalem artichoke powder 8 g/L, peptone 8 g/L, extractum carnis 10 g/L, powder chitin 10 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L.
Condition of enzyme production: under 1L fermentor tank system, inoculum size 5%, initial pH:6, temperature: 37 DEG C, fermentation period: 96 h, rotating speed: 200 rpm, air flow: 1 vvm.
Under above implementation condition, ferment, and carry out enzyme activity determination, method therefor is with embodiment 3.
The crude enzyme liquid enzyme work of gained can reach 0.78 U/mL.
embodiment 6: the application that the present embodiment explanation bacterial strain of the present invention produces chitinase enzymolysis chitin produces 2-acetylamino-2-deoxy-D-glucose
Chitinase that bacterial strain of the present invention produces degrade chitin rapidly, wherein, the product yield of 0.5% concentration tobacco brown spot pathogen 2 h of degrading reaches 86.16%, the product yield of 100 mesh sieve powder chitin 12 h of degrading reaches 65.36%, and degraded product purity is high, enzymolysis product main component through liquid phase and mass spectroscopy 0.5% tobacco brown spot pathogen is 2-acetylamino-2-deoxy-D-glucose, without chitobiose (seeing accompanying drawing 2,4).
embodiment 7: the application (impact of pH value on enzymolysis process) that the present embodiment explanation bacterial strain of the present invention produces chitinase enzymolysis chitin produces 2-acetylamino-2-deoxy-D-glucose.
(1) preparation of thick enzyme:
Centrifugal fermented liquid retains supernatant, adopts saturated ammonium sulphate precipitation classification precipitation target protein and utilizes dialysis method to remove excess salt ion, after acquisition crude enzyme liquid, obtains thick enzyme through vacuum lyophilization.
(2) impact of pH value on enzymolysis process:
To the phosphoric acid buffer that adds different pH values in transformation system, carry out transformation experiment under identical condition respectively, to obtain the affect result of system pH on enzymolysis process, final research shows this enzyme, and suitable enzymolysis pH is that 6.8-7.2(is shown in accompanying drawing 3).
embodiment 8: the application (impact of temperature on enzymolysis process) that the present embodiment explanation bacterial strain of the present invention produces chitinase enzymolysis chitin produces 2-acetylamino-2-deoxy-D-glucose.
Under identical conversion condition (with embodiment 7), variable changes temperature into, and by regulating invert point to find the rising along with temperature, enzyme is lived and continued to rise, and the suitableeest hydrolysis temperature is 40 DEG C, and in the time that temperature is elevated to 45 DEG C, enzyme is lived and can be declined rapidly.
embodiment 9: the application (impact of metal ion on enzymolysis process) that the present embodiment explanation bacterial strain of the present invention produces chitinase enzymolysis chitin produces 2-acetylamino-2-deoxy-D-glucose.
Research to this chitinase shows Ca
2+, Mg
2+, Fe
2+enzymolysis process is had to promoter action, is the Ca of 10 mmol/L to adding final concentration in enzymatic hydrolysis system
2+can improve 43% enzyme and live, Mg
2+, Fe
2+improve respectively enzyme and live 37.82% and 33.07%.And Al
3+, Cu
2+, Zn
2+this enzymolysis process of strongly inhibited, to the Al that adds 10 mmol/L in enzymatic hydrolysis system
3+can suppress the enzymolysis process of this enzyme completely, Cu
2+, Zn
2+having suppressed respectively 65.36%, 62.04% enzyme lives.
embodiment 10: the present embodiment explanation bacterial strain of the present invention produces chitinase storage stability
At 4 DEG C of condition storage crude enzyme liquids, every 24 h detect an enzyme work to detect the storage stability of this enzyme.Experiment shows to preserve after 216 h, and this enzyme still keeps 92% enzyme work, has good storage stability.
although in conjunction with specific embodiments particular content of the present invention is had been described in detail, it is not the restriction to this patent protection domain.In claims limited range, the various amendments that those skilled in the art can make without creative work and adjustment are still subject to this patent protection.
Claims (8)
1. the bacterial strain of chitinase is produced in a strain, it is characterized in that, its Classification And Nomenclature is
chitinibactersp. GC72, has been preserved in Chinese Typical Representative culture collection center C CTCC, deposit number: CCTCC NO:M 2014113 on April 1st, 2014.
2. the bacterial strain described in the claims 1 produces the application in chitinase in fermentation.
3. application according to claim 2, is characterized in that, the culture condition of described bacterial strain is glucose 1～4 g/L, peptone 2～10 g/L, yeast extract paste 2～10 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L,
Culture temperature is 20～37 DEG C, 200 rpm.
4. according to the application described in claim 2-3, it is characterized in that, the condition that described strain fermentation produces enzyme is that nutrient media components is jerusalem artichoke powder 1～8 g/L, peptone 2～8 g/L, extractum carnis 2～10 g/L, powder chitin 1～10 g/L, potassium primary phosphate 0.3 g/L, dipotassium hydrogen phosphate 0.7 g/L, magnesium sulfate 0.5 g/L, initial pH:6～8,30 DEG C of leavening temperatures, rotating speed: 200 rpm, air flow: 0.5～1 vvm.
5. utilize the claims 2-4 to produce the application of chitinase enzymolysis chitin production 2-acetylamino-2-deoxy-D-glucose.
6. application according to claim 5, is characterized in that, described enzymatic hydrolysis condition is that hydrolysis temperature is 20～40 DEG C, and preferred hydrolysis temperature is 40 DEG C.
7. application according to claim 5, is characterized in that, described enzymatic hydrolysis condition is that suitable enzymolysis pH value is 6.0～8.0, preferably 6.8-7.2.
8. application according to claim 5, is characterized in that, described enzymatic hydrolysis condition for to add Ca in enzymatic hydrolysis system
2+, Mg
2+, Fe
2+, preferably adding final concentration is 10 mmol/L Ca
2+.
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| CN104388496A (en) * | 2014-12-18 | 2015-03-04 | 南京工业大学 | Method for producing N-acetyl glucosamine by enzymatically degrading chitin |
| CN104531639A (en) * | 2014-12-18 | 2015-04-22 | 南京工业大学 | Bacteriostatic chitin enzymolysis method |
| CN107475273A (en) * | 2017-10-10 | 2017-12-15 | 中国科学院成都生物研究所 | New chitinase P1724 and its application |
| CN111944787A (en) * | 2020-07-30 | 2020-11-17 | 华南理工大学 | Chitinase fused with carbohydrate binding module as well as preparation method and application thereof |
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| CN104388496B (en) * | 2014-12-18 | 2017-12-22 | 南京工业大学 | A kind of method of enzymic degradation chitin production N acetylglucosamines |
| CN104531639B (en) * | 2014-12-18 | 2017-12-22 | 南京工业大学 | A kind of antibacterial chitin enzyme solution |
| CN104388496A (en) * | 2014-12-18 | 2015-03-04 | 南京工业大学 | Method for producing N-acetyl glucosamine by enzymatically degrading chitin |
| CN107475273A (en) * | 2017-10-10 | 2017-12-15 | 中国科学院成都生物研究所 | New chitinase P1724 and its application |
| CN107475273B (en) * | 2017-10-10 | 2019-12-17 | 中国科学院成都生物研究所 | Novel chitinase P1724 and application thereof |
| WO2021148064A1 (en) | 2020-01-22 | 2021-07-29 | Ustav Makromolekularni Chemie Av Cr, V.V.I. | Biodegradable polyurethane foam, biodegradable polyurethane foam-based material for saccharide-cleaving enzyme production, method of synthesis and use thereof |
| CN111944787A (en) * | 2020-07-30 | 2020-11-17 | 华南理工大学 | Chitinase fused with carbohydrate binding module as well as preparation method and application thereof |
| CN111944787B (en) * | 2020-07-30 | 2022-03-29 | 华南理工大学 | Chitinase fused with carbohydrate binding module as well as preparation method and application thereof |
| CN111944789A (en) * | 2020-09-02 | 2020-11-17 | 鲁东大学 | Method for producing chitinase by fermenting chaetomium globosum and application thereof |
| CN111944789B (en) * | 2020-09-02 | 2021-11-30 | 鲁东大学 | Method for producing chitinase by fermenting chaetomium globosum and application thereof |
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