CN101705221A - Preparation method and application of immobilized acid urease for wine - Google Patents
Preparation method and application of immobilized acid urease for wine Download PDFInfo
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- CN101705221A CN101705221A CN200910233289A CN200910233289A CN101705221A CN 101705221 A CN101705221 A CN 101705221A CN 200910233289 A CN200910233289 A CN 200910233289A CN 200910233289 A CN200910233289 A CN 200910233289A CN 101705221 A CN101705221 A CN 101705221A
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- acid urease
- urea
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- 235000014101 wine Nutrition 0.000 title claims abstract description 74
- 239000002253 acid Substances 0.000 title claims abstract description 57
- 108010046334 Urease Proteins 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 83
- 239000004202 carbamide Substances 0.000 claims abstract description 45
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 17
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 15
- 229920001661 Chitosan Polymers 0.000 claims abstract description 15
- 239000001110 calcium chloride Substances 0.000 claims abstract description 15
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 15
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000000661 sodium alginate Substances 0.000 claims abstract description 14
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 14
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 14
- 230000008569 process Effects 0.000 claims abstract description 6
- 235000019991 rice wine Nutrition 0.000 claims description 23
- 108010091086 Recombinases Proteins 0.000 claims description 16
- 102000018120 Recombinases Human genes 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000008367 deionised water Substances 0.000 claims description 12
- 229910021641 deionized water Inorganic materials 0.000 claims description 12
- 238000013019 agitation Methods 0.000 claims description 10
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 9
- 238000003760 magnetic stirring Methods 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 6
- 241000305071 Enterobacterales Species 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 238000005516 engineering process Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 230000009466 transformation Effects 0.000 claims description 5
- 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 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 230000009471 action Effects 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 241000147019 Enterobacter sp. Species 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 claims description 2
- 230000004151 fermentation Effects 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- RRIWRJBSCGCBID-UHFFFAOYSA-L nickel sulfate hexahydrate Chemical compound O.O.O.O.O.O.[Ni+2].[O-]S([O-])(=O)=O RRIWRJBSCGCBID-UHFFFAOYSA-L 0.000 claims description 2
- 229940116202 nickel sulfate hexahydrate Drugs 0.000 claims description 2
- 238000005057 refrigeration Methods 0.000 claims description 2
- 150000003467 sulfuric acid derivatives Chemical class 0.000 claims description 2
- 238000013016 damping Methods 0.000 claims 2
- 239000012530 fluid Substances 0.000 claims 2
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims 2
- 239000000796 flavoring agent Substances 0.000 abstract description 3
- 235000019634 flavors Nutrition 0.000 abstract description 3
- 239000000654 additive Substances 0.000 abstract description 2
- 230000000996 additive effect Effects 0.000 abstract description 2
- 241000588914 Enterobacter Species 0.000 abstract 1
- 238000004132 cross linking Methods 0.000 abstract 1
- 238000001179 sorption measurement Methods 0.000 abstract 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 15
- 230000000694 effects Effects 0.000 description 11
- 239000000243 solution Substances 0.000 description 9
- 238000013459 approach Methods 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 238000012856 packing Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000002798 spectrophotometry method Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 2
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000000711 cancerogenic effect Effects 0.000 description 2
- 231100000315 carcinogenic Toxicity 0.000 description 2
- 238000005352 clarification Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
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- 238000002156 mixing Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- VRZJGENLTNRAIG-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]iminonaphthalen-1-one Chemical compound C1=CC(N(C)C)=CC=C1N=C1C2=CC=CC=C2C(=O)C=C1 VRZJGENLTNRAIG-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000013124 brewing process Methods 0.000 description 1
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- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- FSEUPUDHEBLWJY-HWKANZROSA-N diacetylmonoxime Chemical compound CC(=O)C(\C)=N\O FSEUPUDHEBLWJY-HWKANZROSA-N 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
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- 235000015097 nutrients Nutrition 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
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- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- 210000001541 thymus gland Anatomy 0.000 description 1
- -1 urethane ester Chemical class 0.000 description 1
Images
Abstract
A preparation method and application of immobilized acid urease for wine belong to the technical fields of enzyme preparation and brewing additive. The preparation method comprises the steps that purified free enzyme separated from enterobacter 9043C12 is subjected to sodium alginate and calcium chloride embedding, chitosan adsorption and trace glutaraldehyde crosslinking to obtain the immobilized acid urease for the wine; the optimal temperature of the immobilized acid urease for the wine is 40 DEG C, optimal pH thereof is 4, and storage half-life period is 71 days; and the invention also relates to the application of the immobilized acid urease for the wine, after yellow wine is added with the immobilized acid urease for the wine, urea removal rate of the yellow wine can reach about 80 percent, and after the immobilized acid urease for the wine is continuously used for 21 times, the urea removal rate can reach 51.6 percent. The invention can realize repeated use of the immobilized acid urease for the wine, without changing the process conditions of the original brewed wine or the flavor of the wine, thus being a more ideal method for removing urea in the wine.
Description
Technical field
A kind of preparation method of immobilized acid urease for wine and application specifically will be from enterobacteria 9043C
12The resolvase of middle separation and purification, through sodium alginate and calcium chloride embedding, chitosan absorption, trace glutaraldehyde is crosslinked, gets immobilized acid urease for wine, belongs to zymin, wine brewing additive technology field.
Background technology
Urethanum (or claim urethane, be called for short EC), 2005 by international cancer research institution (International Agency for Research on Cancer IARC) puts 2B (people is had carcinogenic suspicion material) group under.Its mechanism of carcinogenesis has 3 kinds of approach as can be known by the research to the pathways metabolism of urethanum in the body: first surpasses 90% urethanum can be broken down into (this approach are avirulent) such as ethanol, ammonia and carbohydrate; Second about 0.5% urethanum is oxidized to the DNA addition polymer by Cytochrome P450, causes the dna double chain to destroy, and can cause canceration; The third approach is comparatively general, and promptly about 0.1% urethanum is oxidized to N-hydroxyl-urethanum by Cytochrome P450, and the latter can induce Cu
2+The dna damage of regulation and control, this damage pilosity is born on the residue of thymus pyrimidine (T) and cytosine(Cyt) (C).Great majority think that the third approach is the main carcinogenic approach of urethanum.
Both at home and abroad to urethanum in the wine studies show that the main path that urethanum forms in the wine is the urea approach, the urethanum in China's yellow rice wine and the Japanese sake 90% or more is to derive from that contained urea and alcoholic acid react in the wine:
H
2NCONH
2+C
2H
5OH→C
2H
5OCONH
2+NH
3
And the main source of urea is in the wine: produced by microbial metabolism, i.e. contained urea in the nutrient solution composition of the urea that is produced by the arginine metabolism and raw and auxiliary material and brewing process.
After having studied the factor that influences urethanum formation, find that urea concentration, alcohol concn etc. is topmost factor.The various countries scholars have carried out deep research to the method that reduces urethanum in the wine, by the urea content in removal or the reduction wine, can reach the purpose of control urethane ester content.
In wine, add acid urease for wine and can reduce urea content, but resolvase is unstable in solution, easily sex change and inactivation, reaction back enzyme is difficult to Separation and Recovery, can't reuse, utilize immobilized enzyme can avoid above deficiency, enlarge the use range and the access times of enzyme.Resolvase is made immobilized enzyme, in wine, add immobilized acid urease for wine and do not influence the yellow rice wine normal production process, blending, in the process of clarification wine, add immobilized acid urease for wine, just can reduce urea content, and immobilized acid urease for wine is easy to separate with wine, can reuse, reduce the consumption of acid urease.
China is the country of year liquor output maximum in the world, and very long wine brewing history is arranged, and not only output is big, and wide in variety, and many products are enjoyed higher reputation in the world.The shao-hsing rice wine of China has market widely in Japan and South East Asia, but in 1987, Japan relevant department proposes urethanum content overproof (greater than 100 μ g/L) in the shao-hsing rice wine, in recent years, on the shao-hsing rice wine export trade, run into the problem of urethanum again, in order to reduce the content of urethanum in the wine, shao-hsing rice wine group still needs from Japanese import acid urease for wine so far.Therefore, for people health, also in order more to export goods and earn foreign currency, the acid urease for wine of research and production China oneself has become the task of top priority.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of immobilized acid urease for wine will be from enterobacteria 9043C
12The resolvase of middle separation and purification, through sodium alginate and calcium chloride embedding, chitosan absorption, trace glutaraldehyde is crosslinked, gets immobilized acid urease for wine, and the optimum temperuture of this immobilization acid urease is 40 ℃, and optimal pH is 4, and storing the transformation period is 71 days; The invention still further relates to the application of this immobilized acid urease for wine, in yellow rice wine, add this immobilized acid urease for wine after the urea clearance can reach about 80%, use 21 times continuously after, the urea clearance still can reach 51.6%.The present invention can not change the processing condition of former brewing wine, does not change the local flavor of wine, and immobilized acid urease for wine can reuse, be at present comparatively ideal remove the method for urea in the wine.
Technical scheme of the present invention: a kind of preparation method of immobilized acid urease for wine, will be from enterobacteria 9043C
12The resolvase of middle separation and purification, through sodium alginate and calcium chloride embedding, chitosan absorption, trace glutaraldehyde is crosslinked, gets immobilized acid urease for wine, and its technology is:
A, resolvase extract:
(1) bacterial classification: adopt enterobacteria (Enterobacter sp.) 9043C
12Chinese patent ZL200610096635.1 is open.
(2) seed culture:
Seed culture medium is formed: seed culture medium is in g/L: glucose 20, peptone 10, extractum carnis 10, yeast extract paste 5, KH
2PO
42, NaCl5, NaAc 2, urea 5, pH7.0 (transferring pH with NaOH);
Culture condition: 5%, 35 ℃ of constant temperature of inoculum size leaves standstill cultivates 16～18h;
(3) fermentation culture:
Fermention medium is formed: in g/L: glucose 20, peptone 10, extractum carnis 5, yeast extract paste 5, KH
2PO
42, NaCl 5, and NaAc 2, urea 5, four water manganous sulfates 0.05, nickel sulfate hexahydrate 0.05, pH5.5 (HCl transfers pH);
The fermented liquid culture condition: 6%, 35 ℃ of constant temperature of inoculum size leaves standstill cultivates 28～32h;
(4) collect thalline: behind fermented liquid 8000r/min, 4 ℃ of centrifugal 10min, abandon supernatant, with twice of deionized water wash of thalline, citric acid one sodium citrate buffer solution of pH5.5 washs once, and the every liter of centrifugal back of fermented liquid gained thalline redissolves in the 35mL of pH5.5 citric acid one sodium citrate buffer solution;
(5) ultrasonic disruption: ultrasonic power 300w, broken time 10min, treatment capacity 35mL; Broken liquid in 4 ℃ of centrifugal 20min of following 13000r/min, is collected supernatant liquor, be resolvase enzyme liquid;
B, immobilization process:
(1) sodium alginate is dissolved in the resolvase enzyme liquid, the mass/volume concentration of control sodium alginate reaches 2%, fully stirs evenly, and 4 ℃ of refrigerations are left standstill to no bubble;
(2) dropwise splash in the solution that is dissolved with mass/volume concentration 0.25% chitosan and 5% calcium chloride of its 5 times of volumes with the resolvase enzyme liquid that contains sodium alginate of syringe step (1), magnetic agitation 3h, deionized water wash is removed free chitosan and calcium chloride, drain, get immobilized spherule;
(3) step (2) gained immobilized spherule is placed volumetric concentration 0.1% glutaraldehyde, immobilized spherule quality/glutaraldehyde volume is controlled to be 2g/mL, behind the microwave 3min, place magnetic agitation 2.5h on the magnetic stirring apparatus, deionized water wash is removed free glutaraldehyde, drains, refrigerate standby, immobilized acid urease for wine.
The application of the immobilized acid urease for wine of preparation: the optimum temperuture of this immobilization acid urease is 40 ℃, and optimal pH is 4, and storing the transformation period is 71 days;
Add this immobilized acid urease for wine in yellow rice wine after, enzyme concentration: immobilized enzyme weight/yellow rice wine volume is 100g/L, under dynamic condition, 35 ℃ of temperature, action time, 18h removed in the wine sample 80% urea, after using 21 times continuously, the urea clearance reaches 51.6%.
The character of this immobilized acid urease for wine: optimum temperuture, optimal pH and store determining of transformation period:
In 25-85 ℃ of scope, every 5 ℃, measure enzyme activity respectively, be 100% with high enzymatic activity, calculate relative enzyme activity, optimum temperuture is 40 ℃.
Measuring enzyme respectively and live under condition of different pH, is 100% with high enzymatic activity, calculates relative enzyme activity, and optimal pH is 4.
The immobilization acid urease is put in 4 ℃ of refrigerators, measured the enzyme of immobilization acid urease every three days and live, storing the transformation period is 71 days.
The application of this immobilized acid urease for wine:
(1) under dynamic condition, about 35 ℃ of temperature, enzyme concentration 100g/L (immobilized enzyme weight/yellow rice wine volume), 18 hours action time, about 80% urea in the removal wine sample.
(2) 50g immobilization acid urease is placed pillar, under the drive of constant flow pump, the 80mL yellow rice wine is crossed pillar with the velocity flow of 0.5mL/min, yellow rice wine is measured its urea concentration after the circulation primary in pillar, calculate the urea clearance, using the urea clearance first is 83.4%, and then adds the yellow rice wine revision test of 80mL, repeat 21 times after the urea clearance be 51.6%.
The enzyme activity determination method
Resolvase measuring method alive: adopt indophenol blue reaction (Berthelot reaction) colorimetry.
The enzyme activity definition: at normal pressure, 37 ℃, under the pH5.5 condition, per minute bottom exploded deposits yields 1 μ mol ammonia is an enzyme activity unit.
Immobilized enzyme measuring method alive:
The active mensuration of immobilized acid urease for wine is to measure the activity of supernatant liquor enzyme after the activity of immobilization preferment and the immobilization, calculates activity of the immobilized enzyme then.
Activity of the immobilized enzyme=(activity of supernatant liquor enzyme after the activity-immobilization of immobilization preferment)
The measuring method of urea content: Diacetylmonoxime method.
Determining the protein quantity method: Xylene Brilliant Cyanine G G-250 method.
The mensuration of glutaraldehyde content: ultraviolet spectrophotometry.
Beneficial effect of the present invention: the extracting method of resolvase can replace with industrial process, and the making processes of immobilized enzyme can be made of machine and be replaced, and realizes suitability for industrialized production.This immobilization acid urease can be stablized in wine and plays a role, urea clearance height, in yellow rice wine, add immobilized acid urease for wine after the urea clearance can reach about 80%, and immobilized acid urease for wine is convenient to reclaim, and can reuse 21 times.The present invention can not change the processing condition of former brewing wine, does not change the local flavor of wine, is blending, and in the process of clarification wine, adds immobilized acid urease for wine, just can reduce urea content, is the method that present comparatively ideal is removed urea in the wine.
Description of drawings
Fig. 1 immobilized acid urease for wine preparation technology synoptic diagram.
Embodiment
Embodiment 1: the 2g sodium alginate is dissolved in the free acid urease enzyme liquid of 100mL, fully stir with glass stick, stir evenly and be placed on 4 ℃ of refrigerator and cooled and hide to the solution till the no bubble, dropwise splashing into 500mL with syringe is dissolved with in the solution of 0.25% chitosan and 5% calcium chloride, place magnetic agitation 3h on the magnetic stirring apparatus, use the deionized water thorough washing, remove free chitosan and calcium chloride, drain, immobilized spherule is placed 0.1% glutaraldehyde, immobilized spherule quality/glutaraldehyde volume is controlled to be 2g/mL, behind the microwave 3min, place that magnetic agitation 2.5h (covers with opaque container on the magnetic stirring apparatus, in case glutaraldehyde is seen phototropic), deionized water wash is removed free glutaraldehyde (ultraviolet spectrophotometry detection), drains, refrigerate standby, get immobilized acid urease for wine 50.31g, the flow-through appt of packing into is at internal diameter 2.5cm, in the pillar of long 20cm, the 50.31g immobilized acid urease for wine of packing into, the adjusting flow velocity is 0.5mL/min, makes the yellow rice wine of 200mL flow through pillar, urea content in the mensuration yellow rice wine of each circulation back, calculate the urea clearance, the circulation 3 times after urea content reduce to 4.82mg/L from 23.9mg/L, the urea clearance is 79.8%.
Embodiment 2: the 3.6g sodium alginate is dissolved in the free acid urease enzyme liquid of 180mL, fully stir with glass stick, stir evenly and be placed on 4 ℃ of refrigerator and cooled and hide to the solution till the no bubble, dropwise splashing into 900mL with syringe is dissolved with in the solution of 0.25% chitosan and 5% calcium chloride, place magnetic agitation 3h on the magnetic stirring apparatus, use the deionized water thorough washing, remove free chitosan and calcium chloride, drain, immobilized spherule is placed 0.1% glutaraldehyde, immobilized spherule quality/glutaraldehyde volume is controlled to be 2g/mL, behind the microwave 3min, place that magnetic agitation 2.5h (covers with opaque container on the magnetic stirring apparatus, in case glutaraldehyde is seen phototropic), deionized water wash is removed free glutaraldehyde (ultraviolet spectrophotometry detection), drains, refrigerate standby, get immobilized acid urease for wine 89.58g, the flow-through appt of packing into is at internal diameter 3.5cm, in the pillar of long 30cm, the 89.58g immobilized acid urease for wine of packing into, the adjusting flow velocity is 0.5mL/min, makes the yellow rice wine of 500mL flow through pillar, urea content in the mensuration yellow rice wine of each circulation back, calculate the urea clearance, the circulation 3 times after urea content reduce to 3.96mg/L from 24.6mg/L, the urea clearance is 83.9%.
Embodiment 3: the 2g sodium alginate is dissolved in the free acid urease enzyme liquid of 100mL, fully stir with glass stick, stir evenly and be placed on 4 ℃ of refrigerator and cooled and hide to the solution till the no bubble, dropwise splashing into 500mL with syringe is dissolved with in the solution of 0.25% chitosan and 5% calcium chloride, place magnetic agitation 3h on the magnetic stirring apparatus, use the deionized water thorough washing, remove free chitosan and calcium chloride, drain, immobilized spherule is placed 0.1% glutaraldehyde, immobilized spherule quality/glutaraldehyde volume is controlled to be 2g/mL, behind the microwave 3min, place that magnetic agitation 2.5h (covers with opaque container on the magnetic stirring apparatus, in case glutaraldehyde is seen phototropic), deionized water wash, remove free glutaraldehyde (ultraviolet spectrophotometry detection), drain, refrigerate standby, get immobilized acid urease for wine 50.56g, the flow-through appt of packing into is at internal diameter 2.5cm, in the pillar of long 20cm, the 50.56g immobilized acid urease for wine of packing into, make the 80mL yellow rice wine cross pillar with the velocity flow of 0.5mL/min, yellow rice wine is measured its urea concentration after the circulation primary in pillar, calculate its urea clearance, using the urea clearance first is 83.4%, and then adds the yellow rice wine revision test of 80mL.The urea clearance is 51.6% after repeating 21 times.
Claims (2)
1. the preparation method of an immobilized acid urease for wine is characterized by: will be from enterobacteria 9043C
12The resolvase of middle separation and purification, through sodium alginate and calcium chloride embedding, chitosan absorption, trace glutaraldehyde is crosslinked, gets immobilized acid urease for wine, and its technology is:
A, resolvase extract:
(1) bacterial classification: adopt enterobacteria (Enterobacter sp.) 9043C
12
(2) seed culture:
Seed culture medium is formed: seed culture medium is in g/L: glucose 20, peptone 10, extractum carnis 10, yeast extract paste 5, KH
2PO
42, NaCl 5, and NaAc 2, urea 5, pH7.0;
Culture condition: 5%, 35 ℃ of constant temperature of inoculum size leaves standstill cultivates 16～18h;
(3) fermentation culture:
Fermention medium is formed: in g/L: glucose 20, peptone 10, extractum carnis 5, yeast extract paste 5, KH
2PO
42, NaCl 5, and NaAc 2, urea 5, four water manganous sulfates 0.05, nickel sulfate hexahydrate 0.05, pH5.5;
The fermented liquid culture condition: 6%, 35 ℃ of constant temperature of inoculum size leaves standstill cultivates 28～32h;
(4) collect thalline: behind fermented liquid 8000r/min, 4 ℃ of centrifugal 10min, abandon supernatant, with twice of deionized water wash of thalline, the citric acid-sodium citrate damping fluid of pH5.5 washs once, and the every liter of centrifugal back of fermented liquid gained thalline redissolves in the 35mL of pH5.5 citric acid-sodium citrate damping fluid;
(5) ultrasonic disruption: ultrasonic power 300w, broken time 10min, treatment capacity 35mL; Broken liquid in 4 ℃ of centrifugal 20min of following 13000r/min, is collected supernatant liquor, be resolvase enzyme liquid;
B, immobilization process:
(1) sodium alginate is dissolved in the resolvase enzyme liquid, the mass/volume concentration of control sodium alginate reaches 2%, fully stirs evenly, and 4 ℃ of refrigerations are left standstill to no bubble;
(2) dropwise splash in the solution that is dissolved with mass/volume concentration 0.25% chitosan and 5% calcium chloride of its 5 times of volumes with the resolvase enzyme liquid that contains sodium alginate of syringe step (1), magnetic agitation 3h, deionized water wash is removed free chitosan and calcium chloride, drain, get immobilized spherule;
(3) step (2) gained immobilized spherule is placed volumetric concentration 0.1% glutaraldehyde, immobilized spherule quality/glutaraldehyde volume is controlled to be 2g/mL, behind the microwave 3min, place magnetic agitation 2.5h on the magnetic stirring apparatus, deionized water wash is removed free glutaraldehyde, drains, refrigerate standby, immobilized acid urease for wine.
2. with the application of the immobilized acid urease for wine of claim 1 method preparation: the optimum temperuture that it is characterized in that this immobilization acid urease is 40 ℃, and optimal pH is 4, and storing the transformation period is 71 days;
Add this immobilized acid urease for wine in yellow rice wine after, enzyme concentration: immobilized enzyme weight/yellow rice wine volume is 100g/L, under dynamic condition, 35 ℃ of temperature, action time, 18h removed in the wine sample 80% urea, after using 21 times continuously, the urea clearance reaches 51.6%.
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