CN109652390A - A kind of marine low temperature glucose oxidase and its application - Google Patents
A kind of marine low temperature glucose oxidase and its application Download PDFInfo
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- 235000019420 glucose oxidase Nutrition 0.000 title claims abstract description 46
- 108010015776 Glucose oxidase Proteins 0.000 title claims abstract description 45
- 239000004366 Glucose oxidase Substances 0.000 title claims abstract description 45
- 229940116332 glucose oxidase Drugs 0.000 title claims abstract description 45
- 102000004190 Enzymes Human genes 0.000 claims abstract description 51
- 108090000790 Enzymes Proteins 0.000 claims abstract description 51
- 229940088598 enzyme Drugs 0.000 claims abstract description 51
- 241000287828 Gallus gallus Species 0.000 claims abstract description 12
- 241000598399 Basidioascus sp. Species 0.000 claims abstract description 8
- 239000000872 buffer Substances 0.000 claims abstract description 6
- 238000000855 fermentation Methods 0.000 claims description 9
- 230000004151 fermentation Effects 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 7
- 238000000108 ultra-filtration Methods 0.000 claims description 7
- 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 5
- 239000008103 glucose Substances 0.000 claims description 5
- 235000019733 Fish meal Nutrition 0.000 claims description 4
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 4
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 4
- 235000005822 corn Nutrition 0.000 claims description 4
- 239000004467 fishmeal Substances 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 235000015099 wheat brans Nutrition 0.000 claims description 4
- 235000019750 Crude protein Nutrition 0.000 claims description 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 229920005654 Sephadex Polymers 0.000 claims description 3
- 239000012507 Sephadex™ Substances 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000013535 sea water Substances 0.000 claims description 3
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 3
- 229920001503 Glucan Polymers 0.000 claims description 2
- 238000012870 ammonium sulfate precipitation Methods 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 229920002307 Dextran Polymers 0.000 claims 1
- 238000004587 chromatography analysis Methods 0.000 claims 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 26
- 229910021645 metal ion Inorganic materials 0.000 abstract description 9
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 abstract description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 abstract description 4
- 208000035240 Disease Resistance Diseases 0.000 abstract description 3
- 239000000654 additive Substances 0.000 abstract description 3
- 230000000996 additive effect Effects 0.000 abstract description 3
- 239000006174 pH buffer Substances 0.000 abstract description 3
- 230000003115 biocidal effect Effects 0.000 abstract description 2
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 abstract description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 abstract description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 abstract description 2
- 230000000050 nutritive effect Effects 0.000 abstract description 2
- 230000000717 retained effect Effects 0.000 abstract description 2
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 239000011592 zinc chloride Substances 0.000 abstract description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 19
- 239000000758 substrate Substances 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 235000013330 chicken meat Nutrition 0.000 description 6
- 230000002255 enzymatic effect Effects 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229940036811 bone meal Drugs 0.000 description 2
- 239000002374 bone meal Substances 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
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- 210000000936 intestine Anatomy 0.000 description 2
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- 239000002609 medium Substances 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 235000013594 poultry meat Nutrition 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- JRBJSXQPQWSCCF-UHFFFAOYSA-N 3,3'-Dimethoxybenzidine Chemical compound C1=C(N)C(OC)=CC(C=2C=C(OC)C(N)=CC=2)=C1 JRBJSXQPQWSCCF-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000003643 Callosities Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 240000006927 Foeniculum vulgare Species 0.000 description 1
- 235000004204 Foeniculum vulgare Nutrition 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
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- 230000035622 drinking Effects 0.000 description 1
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- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000008303 genetic mechanism Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 231100000001 growth retardation Toxicity 0.000 description 1
- 230000037219 healthy weight Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000002636 mycotoxin Substances 0.000 description 1
- 235000021590 normal diet Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000011787 zinc oxide Substances 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
- A23K10/22—Animal feeding-stuffs from material of animal origin from fish
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
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- 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
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Abstract
The invention discloses a kind of marine low temperature glucose oxidase and its applications.Marine low temperature glucose oxidase provided by the invention source Yu Haiyang basidiomycetes Basidioascus sp.WYQ 23, deposit number is CGMCC No.17180, enzyme activity is high under cryogenic for marine low temperature glucose oxidase of the present invention, optimum temperature is 20 DEG C, and 4% enzyme activity is thrown away when handling 60min for 80 DEG C；Optimal pH is 5.6, and enzyme activity reduces rapidly when higher than 4.5 or 6.5, and 76.3% enzyme activity is retained when after the buffer of pH9 handling 60min, higher to tolerance under different pH buffer conditions.The metal ion for having humidification to marine low temperature glucose oxidase of the present invention is MgCl2, KCl (see attached drawing 5)；Having the metal ion compared with high inhibition effect to marine low temperature glucose oxidase of the present invention is AgNO3、ZnCl2、FeSO4.It is applied to and when chicken feed addictive, the daily gain and disease resistance of chicken can be effectively improved, and reduce the death rate, antibiotic can be replaced, its nutritive value is improved, be a kind of green additive having no toxic side effect.
Description
Technical field
As the application of feed addictive the present invention relates to a kind of marine low temperature glucolase and its in fowl raising.
Background technique
The application field of glucose oxidase is mostly in middle low temperature environment, such as feed addition, food fresh keeping, cold bleaching
Deng.Low temperature glucose oxidase has broader application range, high enzymatic activity and high catalytic efficiency, can reduce and be produced into
This, improves production efficiency, improves product quality, has broader industrial development value and the market advantage.
Marine environment be have the characteristics that with high salt, high pressure, low temperature, low illumination, oligotrophic extreme environment, and its microorganism
It is resourceful.Ecological environment complicated and changeable and genetic mechanism assign the unique mechanism of action of Enzymes from Marine Microorganisms, therefore ocean
Microorganism is increasingly becoming the important sources of novel enzyme preparation.The cold-adapted enzyme of ocean microorganism, tolerant enzyme etc. are tied both at home and abroad
The research of the extreme enzyme of structure and novel functions gradually increases, and haves laid a good foundation for the development of marine low temperature enzyme research field.
The Ministry of Agriculture in 1999 by GOD be set to 12 kinds allow using one of feed enzyme preparation additive.GOD, which has to destroy, to raise
Mycotoxin in material, extend the feed shelf-life, improve feed quality, improve feed nutrient digestibility, reduction or substitution zinc oxide,
The effects of probiotics and substitute antibiotics, is adjusted animal and bird intestines pH, inhibits harmful bacteria growth in animal and bird intestines, maintains enterobacteriaceae
Group's balance, improve gastrointestinal tract environment, reduce diarrhea occurs, protection intestinal epithelial cell is complete, remove in livestock and poultry body it is extra from
By base, reduce stress damage, reduce livestock and poultry hepar damnification, promote growth, reduce the performances such as the death rate, it has also become researcher and
Breeding enterprise focus of attention.
Summary of the invention
To solve the above problems, the present invention also provides this present invention provides a kind of marine low temperature glucose oxidase
The preparation method of enzyme and its in fowl raising as the application of feed addictive.
To achieve the above object, the invention adopts the following technical scheme:
A kind of marine low temperature glucose oxidase, the marine low temperature glucose oxidase originate from ocean Basidioascus
Sp.WYQ 23, deposit number are CGMCC No.17180.
The gene for encoding above-mentioned marine low temperature glucose oxidase has the nucleotide sequence as shown in SEQ ID:1,
No. Genbank is BankIt2156862BSeq#1MK061427, which is sequenced by transcript profile and is obtained.
A kind of preparation method of above-mentioned marine low temperature glucose oxidase, the method are specific as follows: will
Basidioascus sp.WYQ 23 is inoculated in fermentation medium, and fermented and cultured obtains fermentation liquid, and 4000r/min is centrifuged 10min
Afterwards in 4 DEG C of ammonium sulfate precipitation precipitates overnights, 12000r/min is centrifuged 30min, and it is poly- to cross Portugal for ultrafiltration after precipitating is redissolved using buffer
Sugared chromatographic column obtains pure enzyme solution.
A kind of preparation method of above-mentioned marine low temperature glucose oxidase, the fermentation liquid culture medium are glucose 40-
60g, peptone 3-4g, CaCO36-10g, MgSO40.6-0.7g, KCl 0.3-0.4g, KH2PO43-4g, NaNO33-4g,
The dissolution of 400mL seawater is added to be settled to 1000mL, pH is naturally, 121 DEG C of sterilizing 20min；The temperature of the fermented and cultured is 15-25
DEG C, revolving speed 160r/min-180r/min, incubation time 36h-48h；The ammonium sulfate concentrations are 65%；The centrifugal condition is
12000r/min；The ultra-filtration conditions are the super filter tube that interception is 100kDa, 5000r/min ultrafiltration 10min, the buffer
For the PBS of the pH5.6 of 1mol/L, the glucan column chromatographic stuffing is Sephadex G-200.
Above-mentioned marine low temperature glucose oxidase in fowl raising as feed addictive application method be, by corn,
Base-material is formed after crude protein, wheat bran, dregs of beans, fish meal, bone meal, premix, salt mixing, every 100Kg base-material is added after purification
200mL-400mL marine low temperature glucose oxidase, wherein the parts by weight of each raw material are as follows: 55-60 parts of corn, crude protein 16-
19, wheat bran 3-4, dregs of beans 16-21, fish meal 1-3, premix 1, salt 0.3.
Compared with prior art, the invention has the benefit that marine low temperature glucose oxidase of the invention is for the first time
It is prepared in Basidioascus sp.WYQ 23, there is lower optimum temperature and wider temperature tolerance range, most thermophilic
20 DEG C, enzyme activity 0.660U/mL of degree, it is 0.495U/mL that enzyme activity is measured at 0 DEG C, is 88.4% of enzyme activity under optimum temperature；60
DEG C measuring enzyme activity is 0.402U/mL, is 71.2% of enzyme activity under optimum temperature.Marine low temperature glucose oxidase of the invention is answered
For the daily gain and disease resistance of chicken in chicken feed addictive, can be effectively improved, and the death rate is reduced, antibiotic can be replaced,
Its nutritive value is improved, is a kind of green additive having no toxic side effect.
Detailed description of the invention
The measurement of Fig. 1 marine low temperature glucose oxidase optimal reactive temperature of the present invention；
The measurement of Fig. 2 marine low temperature glucose oxidase of the present invention enzyme stability at different temperatures；
The measurement of Fig. 3 marine low temperature glucose oxidase optimal reaction pH of the present invention；
The measurement of Fig. 4 marine low temperature glucose oxidase enzyme stability at different pH of the present invention；
Influence of Fig. 5 metal ion to marine low temperature glucose oxidase of the present invention.
Specific embodiment
The present invention is described in detail below by the drawings and specific embodiments, but is not limited the scope of the invention.Such as without special
Illustrate, experimental method of the present invention is conventional method, and experiment equipment used, material, reagent etc. can be from business ways
Diameter obtains.
Embodiment 1
For enzyme source of the invention in Basidioascus sp.WYQ 23, specific preparation process is as follows:
Step 1: by Basidioascus sp.WYQ 23 be inoculated in fermentation medium (glucose 40g, peptone 4g,
CaCO3 6g、MgSO4 0.7g、KCl 0.3g、KH2PO4 3g、NaNO33g adds the dissolution of 400mL seawater to be settled to 1000mL, and pH is certainly
So), 25 DEG C of cultivation temperature, revolving speed 160r/min, inoculum concentration 10mL, incubation time 38h obtain fermentation liquid；
Step 2: by fermentation liquid at 4 DEG C, the pelleted by centrifugation 10min of 4000r/min, discarding precipitating, supernatant is ocean
Low temperature glucose oxidase crude enzyme liquid；
Step 3: the ammonium sulfate that the crude enzyme liquid that step 2 obtains is added 65% is stayed overnight in 4 DEG C of salt precipitations；
Step 4: the salt precipitation that step 3 the is obtained PBS buffer solution of pH5.6 is redissolved；
Step 5: the solution that step 4 is obtained uses ultrafiltration in the super filter tube that interception is 100kDa, 5000r/min ultrafiltration
10min leaves and takes protein liquid in the pipe of upper layer；
Step 6: the protein liquid that step 5 is obtained crosses Sephadex G-200 chromatographic column in 4 DEG C, detects enzyme activity, collects pure
High enzyme solutions are spent, marine low temperature glucose oxidase as of the present invention.
Embodiment 2
Influence of the temperature to marine low temperature glucose oxidase of the invention:
(1) measurement of optimal reactive temperature takes enzyme solution obtained in the embodiment of the present invention 1, substrate solution respectively at 0 DEG C, 5
DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 70 DEG C, water-bath at a temperature of 80 DEG C
Substrate solution is added enzyme solution reaction, measures enzymatic activity by 10min.Substrate solution is 5% glucose, 0.07g/L neighbour's connection fennel
Amine, 0.1g/L horseradish peroxidase.
(2) under different temperatures enzyme stability measurement, by enzyme in 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C
At a temperature of respectively after water-bath 15min, 30min, 45min, 60min, substrate solution reaction is added in enzyme solution, measures enzymatic activity, with
(1) relevant temperature enzyme activity is 100% in, calculates opposite enzyme activity at each temperature.
The optimum temperature of marine low temperature glucose oxidase catalysis reaction of the present invention is 20 DEG C, and enzyme activity is rapid when being higher than 60 DEG C
It reduces (see attached drawing 1).At different temperatures, the stability of the enzyme is shown, still there is 42.4% enzyme when handling 60min for 50 DEG C
It is living, still there are 4% enzyme activity (see attached drawing 2), its overall visible high stability when handling 60min for 80 DEG C.
Embodiment 3
Influence of the pH to marine low temperature glucose oxidase of the invention:
(1) measurement of optimal reaction pH takes enzyme solution obtained in the embodiment of the present invention 1, substrate solution to distinguish respectively with pH
It is mixed for 3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,9 PBS buffer solution, by enzyme solution after water-bath 10min at 20 DEG C
Substrate solution is added to be reacted, enzymatic activity is measured.Substrate solution is 5% glucose, 0.07g/L dianisidine, 0.1g/L
Horseradish peroxidase.
(2) under difference pH enzyme stability measurement, be respectively 3,4,5,6,7,8,9 PBS buffer solution by enzyme and the pH
Respectively in 20 DEG C of heat preservations 15min, 30min, 45min, 60min after mixing, substrate solution reaction is added in enzyme solution, measures enzyme activity
Property, with pH enzyme activity corresponding in embodiment 2 (1) for 100%, calculate opposite enzyme activity at each temperature.
The optimal pH of marine low temperature glucose oxidase catalysis reaction of the present invention is 5.6, and enzyme activity is fast when being higher than 4.5 or 6.5
Speed reduces (see attached drawing 3).Under different pH buffer conditions, 76.3% enzyme is retained when after the buffer of pH9 handling 60min
(see attached drawing 4) living, it is seen that higher to tolerance under different pH buffer conditions.
Embodiment 4
Influence of the metal ion to marine low temperature glucose oxidase of the invention:
Influence of the different metal ions to enzyme activity, keeps the temperature at 20 DEG C respectively after enzyme is mixed with different metal ions
After 15min, 30min, 45min, 60min, enzyme solution addition substrate solution reaction obtained in the embodiment of the present invention 1 is taken, enzyme is measured
Activity calculates influence of each metal ion to enzyme activity with blank control for 100%.
The metal ion for having humidification to marine low temperature glucose oxidase of the present invention is MgCl2, KCl (see attached drawing 5)；
Having the metal ion compared with high inhibition effect to marine low temperature glucose oxidase of the present invention is AgNO3、ZnCl2、FeSO4。
Embodiment 5
Chicken feed containing marine low temperature glucose oxidase of the present invention: by mass fraction by 57 parts of corns, 17.5 parts it is thick
Base-material is formed after albumen, 3.5 parts of wheat brans, 17 parts of dregs of beans, 2 parts of fish meal, 2 parts of bone meal, 1 part of premix, 0.3 part of salt mixing, often
300mL marine low temperature glucose oxidase after purification is added in 100Kg base-material to obtain the final product.
100 1 Day-old Broiler Chickens are selected, healthy weight, laying rate difference is not significant (P > 0.05), is randomly divided into
2 groups, every group 50.It is wherein fed using the above-mentioned chicken feed containing marine low temperature glucose oxidase of the present invention for the 1st group, is
Sample example, the 2nd group is fed using normal diet (extremely big day is at complete feed), is reference examples；It supports 40 days, each group raises item
Part is identical, free choice feeding and drinking public water supply.Every group of weight and state are observed and recorded, appetite stimulator, growth retardation are regarded as
It falls ill, experimental result is shown in Table 1:
Table 1
| Weight has a net increase of/g | Disease incidence/% | Survival rate/% | |
| Sample example | 2765 | 2 | 98 |
| Reference examples | 2183 | 8 | 92 |
It is added to the chicken feed of marine low temperature glucose oxidase of the present invention it can be seen from 1 result of table, chicken can be improved
Daily gain and disease resistance, and reduce the death rate.
The above, only the invention patent preferred embodiment, but the scope of protection of the patent of the present invention is not limited to this.
Anyone skilled in the art is in the range disclosed in the invention patent, according to the present invention the technical side of patent
Case and its design are subject to equivalent substitution or change, belong to the scope of protection of the patent of the present invention.
Sequence table
<110>University Of Dalian
<120>a kind of marine low temperature glucose oxidase and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2133
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atggttcttc gatcgtctct tgttgcggcc agcgtggcgc ttgggctcgc ggttctgcct 60
gcgcacgcgc agtacaaccg tgacaatggc tacaagcatc agctcatcag gcgccaagcc 120
gtcacgaatg atacgagtca agtcgccggc aagacgttcg actttgtcgt cgtgggcggc 180
ggagtcggcg gtctcgcaat tgcggcgcgg ctatccgaaa actcaaattt cactgtcgct 240
gttattgaag ccggtggaga cggttcggat gtgcacgacc agatttacat tcccggttac 300
tcgtacttga acggtttgac tgtgtctagc tacgactgga actatgagac agtgcagcag 360
caagagatcg actcgacctc caagatcttt actaaccgat ggccgcgcgg caaaggactt 420
ggcggctctg gcgcgattaa cggactcttc tggtgccggg cgagtcaggc cgaatacaac 480
tcgtgggaca cgctcaatcc gggctcgtcg atccactggg gatgggatga aatgcagaag 540
tacatgaagc ggagtgaaac gctcaccccg ccgccgcaga atgactcgga ttacttccac 600
atctcttacg acattaatga ccacggtacg gatggtccta ttcaggcggg ctggccttgg 660
tacttctatg acgctgtcaa ggactggatt cccgcttggg aagagcttgg tctgccgatt 720
ccgactgatg gcgcatccgg caacaatcgc ggtgcggtga tcaccccttc tacactcaac 780
acggtcaaca tgtctcgctc cgaccccaag gccggataca ttgacccgat gccgccgcgc 840
gacaatctcg ttgtactcac gaaccatcaa gtcacgaaga tcctgtggaa gaccgaccgg 900
gacggcaaca acgttgttgc cgatggtgtc gagttcggtc ctgacaacga gaacctgtct 960
caggtcaagg tcaacaagga ggtcattcta tctggcggca ccgtcaatac tccgcagaca 1020
ctccaactgt cgggcatagg accgcaggca ctgcttgaag gcctcggcat ccaggtccaa 1080
gtcgcgcttc caggagttgg gcataatctg caggaccacg cagcgaccgc tgtatacctg 1140
acgacgaacg accagcacac ttgggcggag ctccaagaca agactctcgc cgcagcggag 1200
ctccagaagt ggaagaccga gatgggccgc tccaaatggt ctttcattaa tcaggcgatc 1260
ggttaccctg cgcttgagga catcatgacc gatacggcga cgcggacgag ctggatcaac 1320
ggtgtccaga gtagcttcga caagtacgct tcgactgtca agaacatgta caatgaggac 1380
gatacggtca tcaccggaat gcaggcgcaa tggaacgaga caatgaatta cctgaatggc 1440
gaggttggcc agctcgagat cctcttcacc atgtatggtg gcggtgtcgg tcagatcggt 1500
attcaagtcg cgctgcaaca ccccttctcg cgcggcacga ttctgattaa ctccacgaac 1560
cccttcgacc cgcctcagat tgaccccggg taccttaccc actcagctga catggacatt 1620
ctccgcgccg gtgtgaggtt cgctatcgcg ctttccaaga ctaagtcgct cggctcgtac 1680
atcagcggtc agacatcctt caccgaagat acggatgacg ctatcaacac aggcatccgt 1740
caggatgcgc acacggagta ccaccctatg ggtaccgccg ccatgttgcc gctcgaaaat 1800
ggcggtgtcg tcaatacgtc acttattgtt tacggtactt cgaacgtccg cgtcgttgat 1860
gcttcggtca tgcctctcga aatctccgcg cacttgatgg cgtcgactta cggaatcgct 1920
gagaaggctg ccgacattat caagacgcac tacgcacagg tcgcctctgg ccagaccccc 1980
accgttggtg atcccgacag cggcgcagcg gcgtcgacgt cggcatctgc cggcgcaagc 2040
caagactcgg gcgccatgtc gtctacgacg cgttcgatca tcattggatg tgtcgttggc 2100
gctgccgggc ttgccgtcat cgcggtaagc tga 2133
Claims (6)
1. a kind of marine low temperature glucose oxidase, which is characterized in that the marine low temperature glucose oxidase originates from ocean
Basidioascus sp.WYQ 23, deposit number are CGMCC No.17180.
2. the gene for encoding marine low temperature glucose oxidase described in claim 1 has the nucleotide as shown in SEQ ID:1
Sequence, No. Genbank is BankIt2156862BSeq#1MK061427.
3. a kind of preparation method of marine low temperature glucose oxidase described in claim 1, which is characterized in that the method tool
Body is as follows: Basidioascus sp.WYQ 23 being inoculated in fermentation medium, fermented and cultured obtains fermentation liquid, 4000r/min
It is centrifuged after 10min in 4 DEG C of ammonium sulfate precipitation precipitates overnights, 12000r/min is centrifuged 30min, and precipitating is surpassed after being redissolved using buffer
Filter crosses dextran chromatography column, obtains pure enzyme solution.
4. the preparation method of marine low temperature glucose oxidase as described in claim 1, which is characterized in that the fermentation liquid training
Supporting base is glucose 40-60g/L, peptone 3-4g/L, CaCO36-10g/L, MgSO40.6-0.7g/L, KCl 0.3-0.4g/
L, KH2PO43-4g/L, NaNO33-4g/L, seawater are prepared, and pH is naturally, 121 DEG C of sterilizing 20min；The temperature of the fermented and cultured
It is 15-25 DEG C, revolving speed 160r/min-180r/min, incubation time 36h-48h；The ammonium sulfate concentrations are 65%；The centrifugation
Condition is 12000r/min；The ultra-filtration conditions are the super filter tube that interception is 100kDa, 5000r/min ultrafiltration 10min, described
Buffer is the PBS of the pH5.6 of 1mol/L, and the glucan column chromatographic stuffing is Sephadex G-200.
5. application of the marine low temperature glucose oxidase described in claim 1 in chicken feed.
6. a kind of chicken feed containing marine low temperature glucose oxidase described in claim 1, which is characterized in that the chicken is raised
Material is the 200mL-400mL marine low temperature glucose oxidase being added in every 100Kg base-material after purification；Wherein, the base-material is pressed
The parts by weight of following raw material form: 55-60 parts of corn, 16-19 parts of crude protein, 3-4 parts of wheat bran, 15-19 parts of dregs of beans, fish meal 1-
3 parts, 1 part of premix, 0.3 part of salt.
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| CN110885801A (en) * | 2019-11-25 | 2020-03-17 | 中国海洋大学 | Glucose oxidase M5GOD and coding gene and application thereof |
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