CN102051355A - Preparation method and application of immobilized acidic urease membrane - Google Patents
Preparation method and application of immobilized acidic urease membrane Download PDFInfo
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- CN102051355A CN102051355A CN 201010572018 CN201010572018A CN102051355A CN 102051355 A CN102051355 A CN 102051355A CN 201010572018 CN201010572018 CN 201010572018 CN 201010572018 A CN201010572018 A CN 201010572018A CN 102051355 A CN102051355 A CN 102051355A
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- acid urease
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Abstract
The invention discloses a preparation method and application of an immobilized acidic urease membrane, belonging to the technical field of enzymic preparations and brewing additives. The preparation method comprises the steps of: dissolving acidic urease which is separated and purified from enterobacter 9043C12 in a certain amount of chitosan solution; pouring the solution in a smooth glass plate; naturally dehydrating and drying into a membrane at a temperature of 37DEG C; and crosslinking by using micro amount of glutaraldehyde so as to obtain the immobilized acidic urease membrane, wherein the immobilized acidic urease membrane has the optimum temperature of 40DEG C, the optimum pH of 4.5, the storage half-time of 40d and the enzyme activity recovery of 64.3%. The application of the immobilized acidic urease membrane is that the urea removal rate can achieve 73% after the membrane is added in yellow rice wine and still can achieve 50% after 10-times continuous use. A product of the immobilized acidic urease membrane takes a membrane as a fixed carrier which has a large contact area and can be repeatedly used; and when the immobilized acidic urease membrane is used in the yellow rice wine, both the conditions of the original brewing process and the flavor of the wine are not changed and the urea in the wine can be removed more ideally.
Description
Technical field
The preparation method and the application of immobilization acid urease enzyme membrane are specifically from enterobacteria 9043C
12Middle separation and purification obtains acid urease; Acid urease is dissolved in the certain amount of chitosan solution, pours in the slick sheet glass, 37 ℃ of following seasoning dehydration film forming, trace glutaraldehyde is crosslinked, gets immobilization acid urease enzyme membrane, belongs to zymin, wine brewing additive technology field.
Background technology
Acid urease (acid urease) is the enzyme that urea (urea) can be decomposed into ammonia and carbonic acid gas or volatile salt.This enzyme is distributed widely in the seed of plant, but abundant with content in soybean, the sword bean.Also be present in animal blood, urine and the microbe.Can tolerate sour environment, in the system of pH4.0 ~ 5.5, still have activity.Urea be in the wine by the by product that is produced in the microbial metabolism, be the precursor substance that forms urethanum.
Urethanum (EC) is the product of following of leavened food (as bread, yogurt milk, cheese etc.) and alcoholic beverage (as grape wine, hard cider, Chinese rice wine and Japanese sake etc.).Urethanum is to main the drinking from alcoholic beverage of people's harm.Investigation shows that the urethane ester content is greater than 30 * 10
-6The wine of μ g/L, the people drinks the back and easily suffers from cancer; Urethanum can be surveyed hardly in malt beverage, and on average contains 10 * 10 in the fruit wine brandy
-9~ 73 * 10
-9μ g/L is in yellow rice wine and the pure mellow wine about 20 * 10
-9~ 300 * 10
-9μ g/L, the amount of urethanum is 50 * 10 in most whiskys
-9~ 200 * 10
-9μ g/L is in the food and drink grape wine about 7 * 10
-9~ 12 * 10
-9μ g/L.
After deliberation, urethanum is generated by carbamino compound and ethanol spontaneous reaction:
According to EC content in urea source and urea in the wine and the ethanol synthesis mechanism control wine three approach can be arranged: 1. suitable control fermentation condition, such as temperature, pH value etc.; 2. the yeast of urea is hanged down in seed selection; 3. add an amount of acid urease for wine in the wine after fermentation, with the EC precursor substance urea that is decomposed to form.
More than eliminate in three kinds of methods of EC, the most feasible with the third method especially.This method is not only simple and easy, instant effect, and has following evident characteristic: the flavor characteristics that does not change former wine; Do not change the processing condition of former brewing wine, make production unaffected; The content that reduces EC in the wine can reach about 90%; And in becoming wine, can not detect the existence of active acid urease.
The shao-hsing rice wine of China especially has vast market in Japan in the world.1987, Japanese relevant department proposed, and the EC phenomenon that exceeds standard is arranged in the shao-hsing rice wine, wished that producer takes measures to control the content of EC.At present, wine does not form throughput with urase as yet in China, and the wine that wine brewing needs in producing need spend greatly with urase
The amount foreign exchange is from external import.Therefore, research wine all has profound significance with the development of urase to China's wine-making industry, expansion world market etc.
Free acid urease is because vigor is difficult for keeping, is difficult to reuse and be difficult to shortcomings such as long storage, and its application is subjected to certain restriction.And the immobilization acid urease can be used as a good reactive system, and the enzyme of nature is incorporated on the insoluble solid support material. acid urease stability is strengthened, and can reuse.Enzyme membrane has greatly improved reaction efficiency with the separation function mixing together of the catalysis and the film of enzyme when reducing cost.The natural macromolecular chitosan material is nontoxic in addition, and good hydrophilic property has the natural microenvironment of suitable enzymic catalytic reaction, and cheap, and the source is abundant, can be applicable to food service industry.So this immobilization acid urease enzyme membrane has good prospects for application.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of immobilization acid urease enzyme membrane is from enterobacteria 9043C
12Middle separation and purification is extracted and is obtained acid urease, acid urease is dissolved in the certain amount of chitosan solution, pour in the slick sheet glass, 37 ℃ of following film forming that dehydrate naturally, crosslinked with trace glutaraldehyde, get immobilization acid urease enzyme membrane, the optimum temperuture of this immobilization acid urease is 40 ℃, optimal pH is 4.5, and storing the transformation period is 40 days, and the enzyme rate of recovery alive is 64.3%; The invention still further relates to the application of this immobilization acid urease enzyme membrane, add in yellow rice wine behind this immobilization acid urease enzyme membrane first that the urea clearance can reach 73%, use 10 times continuously after, the urea clearance still can reach 50%.As the fixed carrier, contact area is big, can reuse with film for this acid urease enzyme membrane product, has the incomparable advantage of other carriers; Being applied in the yellow rice wine, can not changing the processing condition of former brewing wine, do not change the local flavor of wine, is the method that present comparatively ideal is removed urea in the wine.
Technical scheme of the present invention: the preparation method of immobilization acid urease enzyme membrane, from enterobacteria 9043C
12Middle separation and purification obtains acid urease, and acid urease is dissolved in the certain amount of chitosan solution, pours in the slick sheet glass, and 37 ℃ dehydrate film forming naturally, and is crosslinked with trace glutaraldehyde, gets immobilization acid urease enzyme membrane, and its technology is:
A. the extraction of acid urease:
(1) bacterial classification: adopt existing enterobacteria (Enterobacter sp.) 9043C in laboratory
12, this bacterial strain is at " Zhejiang Agricultural Univ's journal " 22(5): 493 ~ 498,1996 is open.
(2) seed culture:
Seed culture based component g/L: glucose 20, peptone 10, extractum carnis 10, yeast extract paste 5, KH
2PO
42, NaCl 5, and NaAc 2, and urea 5 is settled to 1L with deionized water, pH7.0;
5%, 35 ℃ of constant temperature of inoculum size leaves standstill cultivates 18 ~ 20h;
(3) fermentation culture:
Fermentation culture based component 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, and nickel sulfate hexahydrate 0.05 is settled to 1L with deionized water, pH5.5;
5%, 35 ℃ of constant temperature of inoculum size leaves standstill cultivates 28 ~ 32 h;
(4) collect thalline: fermented liquid behind centrifugal 10min under 8000r/min, the 4 ℃ of conditions, is abandoned supernatant,
With thalline deionized water wash twice, the citrate buffer solution of pH5.5 washs once, and centrifugal back gained thalline finally is dissolved in the citrate buffer solution of pH5.5, and 1g bacterium mud is dissolved in this citrate buffer solution of 35mL;
(5) high pressure fragmentation: under the 850Pa condition, the each 25mL of treatment capacity, 15min handles 100mL's
Bacterium mud solution, the rate of recovery reaches 80%, is fit to mass-producing and handles; Broken liquid in 4 ℃ of centrifugal 20min of following 14500r/min, is collected supernatant liquor, be required acid urease enzyme liquid;
B, immobilization process:
(1) get in the acetate that the 0.8g chitosan is dissolved in 100mL mass concentration 1%, 4 mL acid urease enzyme liquid be dissolved in wherein, place on the magnetic stirring apparatus, stir evenly deaeration after, filter, with 0.3-0.5mL filtrate/cm
2Amount be poured on the slick sheet glass, dehydrate film forming under 37 ℃, this film is peeled off after soaking with damping fluid.
(2) film that makes is cut into 4 cm * 4 cm～10 cm * 10 cm square of uniform size, be immersed in the glutaraldehyde solution of mass concentration 0.02%, place magnetic agitation 2h on the magnetic stirring apparatus, deionized water wash, remove free glutaraldehyde, drain, the gained fixed film is stored in 4 ℃ of refrigerators, and is standby.
The application of the immobilization acid urease enzyme membrane of preparation: the optimum temperuture of this immobilization acid urease is 40 ℃, and optimal pH is 4.5, and storing the transformation period is 40 days, and the enzyme rate of recovery alive is 64.3%;
Add this immobilization acid urease enzyme membrane in yellow rice wine after, add the amount of enzyme membrane: immobilized enzyme volume/yellow rice wine volume is 16 cm
2/ 100mL～100 cm
2/ 100mL, under stirring condition, 37 ℃ of effect 24h can remove in the wine sample 73% urea first, use repeatedly continuously after, the urea clearance reaches 50%.
The character of this immobilized acid urease for wine: optimum temperuture, optimal pH and store determining of transformation period:
In 20-80 ℃ of scope, every 5 ℃, measure enzyme activity respectively, be 100% with high enzymatic activity, meter
Calculate relative enzyme activity, optimum temperuture is 40 ℃.
In pH 3.0-7.5 scope, every 0.5 pH gradient, measure enzyme respectively and live, be 100% with high enzymatic activity, calculate relative enzyme activity, optimal pH is 4.5.
Immobilization acid urease enzyme membrane is put in the citrate buffer solution, preserves in 4 ℃ of refrigerators, measured the enzyme of immobilization acid urease enzyme membrane every three days and live, storing the transformation period is 40 days.
The application of this immobilized acid urease for wine:
The immobilization acid urease enzyme membrane of certain area is placed yellow rice wine, under agitation condition, 37 ℃ of temperature, behind the effect 24h, measure its urea concentration, calculate the urea clearance, using the urea clearance in the yellow rice wine of back first is 73%, then film is taken out with damping fluid and cleans, add the yellow rice wine revision test of 100mL again, repeat 10 times after the urea clearance still can reach 50%.
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, it is an enzyme activity unit that per minute decomposition substrate urea produces 1 μ mol ammonia.
Immobilization acid urease enzyme activity determination method: get 1cm
2In 3% urea soln of the pH5.5 of the citrate buffer solution preparation of film immersion certain volume, behind the insulation 30min, cross its filtrate of leaching in 37 ℃ of constant water bath box, remaining method is identical with the resolvase vitality test.
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 adopts the method for easy mass-producing, and the making processes of immobilization acid urease enzyme membrane can be made of machine and be replaced, and realizes suitability for industrialized production.This immobilization acid urease enzyme membrane can be stablized in wine and plays a role, urea clearance height, in yellow rice wine, add immobilization acid urease enzyme membrane after the urea clearance can reach 73%, and immobilization acid urease enzyme membrane is convenient to reclaim, and can reuse repeatedly.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 immobilization acid urease enzyme membrane, just can reduce urea content, is the method that present comparatively ideal is removed urea in the wine.
Description of drawings
Fig. 1 immobilization acid urease enzyme membrane preparation technology synoptic diagram.
Embodiment
Embodiment 1: the 4mL acid urease is dissolved in 0.8% the chitosan solution of 100mL, fully stirs with glass stick, stir evenly and be placed on 4 ℃ of refrigerator and cooled and hide to the solution till the no bubble, with 0.3mL/cm
2Amount
Pour on the slick sheet glass, 37 ℃ of following drying and forming-films are with peeling off after a small amount of citrate buffer solution immersion, drain, after film is cut into even size box, place 0.02% glutaraldehyde solution, place that magnetic agitation 2h(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, immobilization acid urease enzyme membrane.Get the film of 10 cm * 10 cm sizes, put into the yellow rice wine of 100mL, 37 ℃ act on 24h down, measure urea content in the yellow rice wine, calculate the urea clearance, and urea content is reduced to 6.7mg/L from 24.8mg/L, and the urea clearance reaches 73%.
Embodiment 2: the 4mL acid urease is dissolved in 0.8% the chitosan solution of 100mL, fully stirs with glass stick, stir evenly and be placed on 4 ℃ of refrigerator and cooled and hide to the solution till the no bubble, with 0.5mL/cm
2Amount pour on the slick sheet glass, 37 ℃ of following drying and forming-films are peeled off after soaking with a small amount of citrate buffer solution, drain, after film is cut into even size box, place 0.02% glutaraldehyde solution, place that magnetic agitation 2h(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, immobilization acid urease enzyme membrane.Get the film of 4 cm * 4 cm sizes, put into the yellow rice wine of 100mL, 37 ℃ act on 24h down, measure urea content, calculate the urea clearance.Urea content is reduced to 9.6mg/L from 24.8mg/L.Take out film and clean with damping fluid, place the yellow rice wine of 100mL again, urea content in the yellow rice wine is measured in circulation, circulates after 10 times, and the urea clearance also can reach 50%.
Claims (2)
1. the preparation method of an immobilization acid urease enzyme membrane is characterized in that: from enterobacteria 9043C
12Middle separation and purification is extracted and is obtained acid urease; Acid urease is dissolved in the certain amount of chitosan solution, pours in the slick sheet glass, 37 ℃ of following film forming that dehydrate naturally, crosslinked with trace glutaraldehyde, get immobilization acid urease enzyme membrane; Its technology is:
A. the extraction of acid urease:
(1) bacterial classification: adopt existing enterobacteria (Enterobacter sp.) 9043C in laboratory
12;
(2) seed culture: seed culture based component g/L: glucose 20, peptone 10, extractum carnis 10, yeast extract paste 5, KH
2PO
42, NaCl 5, and NaAc 2, and urea 5 is settled to 1L with deionized water, pH7.0;
5%, 35 ℃ of constant temperature of inoculum size leaves standstill cultivates 18 ~ 20h;
(3) fermentation culture: fermentation culture based component 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, and nickel sulfate hexahydrate 0.05 is settled to 1L with deionized water, pH5.5;
5%, 35 ℃ of constant temperature of inoculum size leaves standstill cultivates 28 ~ 32 h;
(4) collect thalline: with fermented liquid behind centrifugal 10min under 8000r/min, the 4 ℃ of conditions, abandon supernatant, with twice of deionized water wash of thalline, the citrate buffer solution washing of pH5.5 once, centrifugal back gained thalline finally is dissolved in the citrate buffer solution of pH5.5, and 1g bacterium mud is dissolved in this citrate buffer solution of 35mL;
(5) high pressure fragmentation: under the 850Pa condition, each treatment capacity 25mL, 15min handles the bacterium mud solution of 100mL; Broken liquid in 4 ℃ of centrifugal 20min of following 14500r/min, is collected supernatant liquor, be required acid urease enzyme liquid;
B. immobilization process:
(1) get in the acetate that the 0.8g chitosan is dissolved in 100mL mass concentration 1%, 4 mL acid urease enzyme liquid be dissolved in wherein, place on the magnetic stirring apparatus, stir evenly deaeration after, double gauze filters, with 0.3-0.5mL filtrate/cm
2Amount be poured on the slick sheet glass, 37 ℃ of dehydration film forming are naturally peeled off after soaking with citrate buffer solution;
(2) film that makes is cut into 4 cm * 4 cm～10 cm * 10 cm square of uniform size, be immersed in the glutaraldehyde solution of mass concentration 0.02%, place magnetic agitation 2h on the magnetic stirring apparatus, deionized water wash, remove free glutaraldehyde, drain, the gained fixed film is stored in 4 ℃ of refrigerators, and is standby.
2. with the application of the immobilization acid urease enzyme membrane of the method for claim 1 preparation: the optimum temperuture that it is characterized in that this immobilization acid urease enzyme membrane is 40 ℃, and optimal pH is 4.5, and storing the transformation period is 40 days;
Add this immobilization acid urease enzyme membrane in yellow rice wine after, add the amount of enzyme membrane: immobilization acid urease enzyme membrane area/yellow rice wine volume is 16 cm
2/ 100mL～100 cm
2/ 100mL, under stirring condition, 37 ℃ of effect 24h can remove in the wine sample 73% urea first, use 10 times continuously after, the urea clearance reaches 50%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114903983A (en) * | 2022-05-23 | 2022-08-16 | 江苏恰瑞生物科技有限公司 | Immobilized uricase particles and preparation method and application thereof |
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| CN1955282A (en) * | 2006-10-12 | 2007-05-02 | 江南大学 | Preparation method and application of fermentation producing wine acid urase using intestinal bacillus |
| US20090186066A1 (en) * | 2000-08-10 | 2009-07-23 | Osfarma S.L. | Method for production of chitosan-based films with enhanced cell adhering capacity, resulting product and applications |
| CN101705221A (en) * | 2009-11-23 | 2010-05-12 | 江南大学 | Preparation method and application of immobilized acid urease for wine |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090186066A1 (en) * | 2000-08-10 | 2009-07-23 | Osfarma S.L. | Method for production of chitosan-based films with enhanced cell adhering capacity, resulting product and applications |
| CN1955282A (en) * | 2006-10-12 | 2007-05-02 | 江南大学 | Preparation method and application of fermentation producing wine acid urase using intestinal bacillus |
| CN101705221A (en) * | 2009-11-23 | 2010-05-12 | 江南大学 | Preparation method and application of immobilized acid urease for wine |
Non-Patent Citations (3)
| Title |
|---|
| 《Enzyme and Microbial Technology》 20000430 Senay Akkus et al. Immobilization of catalase on chitosan film 497-501 1-2 第26卷, 第7期 2 * |
| 《中国优秀硕士学位论文全文数据库 基础科学辑》 20100515 王松华 一种酸性脲酶的纯化及产生菌的鉴定与分析 A006-85 1-2 , 第5期 2 * |
| 《食品与生物技术学报》 20100531 吴召慧 等 包埋吸附法固定化酒用酸性脲酶的研究 437-441 1-2 第29卷, 第3期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114903983A (en) * | 2022-05-23 | 2022-08-16 | 江苏恰瑞生物科技有限公司 | Immobilized uricase particles and preparation method and application thereof |
| CN114903983B (en) * | 2022-05-23 | 2023-08-15 | 江苏恰瑞生物科技有限公司 | Immobilized uricase particle, and preparation method and application thereof |
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