CN102242109A - Method for keeping stability of immobilized acid urease membrane - Google Patents
Method for keeping stability of immobilized acid urease membrane Download PDFInfo
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- CN102242109A CN102242109A CN2011101093989A CN201110109398A CN102242109A CN 102242109 A CN102242109 A CN 102242109A CN 2011101093989 A CN2011101093989 A CN 2011101093989A CN 201110109398 A CN201110109398 A CN 201110109398A CN 102242109 A CN102242109 A CN 102242109A
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- acid urease
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Abstract
The invention discloses a method for keeping the stability of an immobilized acid urease membrane, belonging to the technical fields of enzyme preparations, food additives and biochemical engineering. The method disclosed by the invention comprises the following steps of: adding different stabilizers into acid urease, selecting sucrose and glycerol with good stabilizing effect, and bathing in water for 3 hours at 70 DEG C, wherein the enzyme activity retention rates are respectively 1.34 times and 1.2 times of those without adding the stabilizers; adding the two stabilizers into the immobilized acid urease membrane, wherein the optimal enzyme adding quantity in 100mL of mixed solution is 2mL, meanwhile, the enzyme activity recovery rate without adding the stabilizers is 66.4%, and the enzyme activity recovery rate after the stabilizers are added reaches 82.25%; storing for 60 days at room temperature, wherein the enzyme activity loss without adding the stabilizers is 34.6%, the enzyme activity loss is 21.8% after the sucrose is added, and the enzyme activity loss is 22.4% after the glycerol is added; reacting for 13 batches in a simulated wine solution to enable the enzyme activity to be 53% of the initial enzyme activity; and recycling for 7 times in a yellow wine to enable the enzyme activity to be 52% of the initial enzyme activity. The properties and application of the immobilized acid urease membrane can not be influenced by adding the stabilizers.
Description
Technical field
A kind of method of being maintained fixed acid urease enzyme membrane stability; specifically from several stablizers such as carbohydrate, polyalcohols, inorganic salts; selection is to best stablizer sucrose and the glycerine of acid urease protection effect; sucrose, glycerine are joined respectively in the immobilization acid urease enzyme membrane; investigate aspects such as its unit surface enzyme activity, the enzyme rate of recovery alive, stability in storage, local flavor, polyphenol content variation and operational stability under the room temperature, belong to zymin, foodstuff additive, technical field of biochemical industry.
Background technology
The active function of enzyme depends on complete structure of its molecule and strict structure picture, in drying, low temperature, freeze the space structure that can cause enzyme in molten or the refrigerated storage process repeatedly and destroy, thereby lose its original biological activity, the inactivation of enzyme is the important factor of restriction enzyme preparation industrial production and application, and how improving the stability of enzyme and the inactivation of minimizing enzyme is the focus of biological chemical field research in recent years.At present, the method that improves enzyme stability mainly contains 3 kinds, promptly adds protective material, chemically modified and immobilization and handles.
The immobilization of enzyme (Immobilization of enzymes) is with the enzyme constraint or is limited in certain zone, still can carry out its distinctive catalyzed reaction, and a class technology recyclable and recycling.Compare with resolvase, immobilized enzyme has overcome the deficiency of resolvase again when keeping its efficient single-minded and gentle catalyzed reaction characteristic, improved the stability of enzyme molecular structure, simplified production technique, immobilized enzyme can use aborning repeatedly continuously, has improved the utilization ratio of enzyme.
But; it is the key factor of restriction immobilized enzyme widespread use that enzyme loses vigor in immobilized process; certain density stablizer is added in employing in the enzyme immobilization process; biologically active substances such as enzyme, albumen are played stable provide protection; improve stability; reduce the protoenzyme usage quantity, to the suitability for industrialized production of enzyme with application has important practical significance and economic worth.
Summary of the invention
The objective of the invention is from several stablizers such as carbohydrate, polyalcohols, inorganic salts; selection is to the effective stablizer of acid urease protection; adding sucrose and glycerine are stablizer in immobilization acid urease enzyme membrane; can improve the stability of film; and this method does not influence the application of immobilization acid urease enzyme membrane; do not change the character and the state of film, have a good application prospect.
Technical scheme of the present invention: a kind of method of being maintained fixed acid urease enzyme membrane stability, getting 0.8g chitosan and 0.8g gelatin is dissolved in the acetate of 100mL mass concentration 1%, with 2 mL acid ureases be dissolved in wherein the acid urease mixed solution, in described acid urease mixed solution, add final concentration and be the sucrose of 3g/L or final concentration and be 10% glycerine as stablizer, place on the magnetic stirring apparatus, after stirring evenly deaeration, double gauze filters, with 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; The film that makes is cut into 5 cm * 5 cm square of uniform size, place mass concentration 0.02% glutaraldehyde solution, place magnetic agitation 2h on the magnetic stirring apparatus, cover, in case glutaraldehyde is seen phototropic with opaque container, deionized water wash, remove free glutaraldehyde (ultraviolet spectrophotometry detection), drain, the immobilization acid urease enzyme membrane of the stability that is maintained, gained immobilization acid urease enzyme membrane places room temperature preservation, and the results of regular determination enzyme is lived.
All with the citrate buffer solution preparation of pH5.0,4 ℃ store for future use for described stablizer, the storage liquid of its stablizer.
Acid urease mixed solution behind the interpolation stablizer is after handling 3h under 70 ℃ of water-baths, and with the frozen water cooling, this mixed solution of getting certain volume dilutes 10 times immediately, is used to survey its enzyme and lives.
The immobilization acid urease enzyme membrane that adds stablizer is acted on respectively in 30mL yellow rice wine wine sample or the 30mL simulation wine wine sample, and 37 ℃ of following 100r/min shaking table reaction 12h are one batch, shift out reaction solution, sampling and measuring enzyme activity, urea clearance, polyphenol content; Again add fresh described wine sample solution, survey immobilized enzyme vigor and urea clearance again, so repeatable operation and measure the operational stability of immobilization acid urease enzyme membrane.
A kind of immobilization acid urease of the present invention enzyme membrane Study of Stabilizers; specifically from several stablizers such as carbohydrate, polyalcohols, inorganic salts; selection is to best stablizer sucrose and the glycerine of acid urease protection effect; sucrose, glycerine are joined in the immobilization acid urease enzyme membrane; investigate aspects such as its unit surface enzyme activity, the enzyme rate of recovery alive, stability in storage, local flavor, polyphenol content variation and operational stability under the room temperature, its technology is:
A. acid urease selection of stabilizers:
The acid urease bacterial strain is produced in (1) one strain, its classification called after enterobacter (Enterobacter sp.) 9043C
12, this bacterial strain is at " Zhejiang Agricultural Univ's journal " 22(2): 177 ~ 180, and " Zhejiang Agricultural Univ's journal " 22(5): 493 ~ 498,1996 is open.
(2) stablizer storage liquid is all prepared with the citrate buffer solution of pH5.0, wherein carbohydrate (glucose, sucrose) is formulated as 1 g/L, 2 g/L, 3 g/L, 4 g/L and 5 g/L different concns, polyalcohols (glycerine) be formulated as 5%, 10%, 15%, 20% and the 25%(massfraction) concentration, sorbyl alcohol preparation l g/L, 2 g/L, 3 g/L, 4 g/L and 5 g/L, inorganic salt (sodium-chlor) preparation 10mmol/L, 20mmol/L, 30mmol/L, 40mmol/L and 50mmol/L, 4 ℃ store for future use.With enzyme liquid and several stablizer storage liquid respectively with the volume mixture of 100:1.The enzyme liquid that is added with different sorts, different concns stablizer after handling 3h under 70 ℃ of water-baths, immediately with the frozen water cooling, is got 10 times of the mixed solution dilutions of certain volume, survey its enzyme and live.
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.
(3) compare with the enzyme liquid that does not add stablizer, investigate thermostability respectively, find out the suitableeest stablizer and optimal concentration thereof.
[enzyme before the enzyme work/interpolation stablizer after the interpolation stablizer is handled is handled is lived] * 100%=enzyme retention rate alive
(4) work has good provide protection to the acid urease enzyme for glycerine, sucrose, and glycerol concentration is that 10% o'clock enzyme retention rate of living is 75.5%, is not add 1.2 times of stablizer.The enzyme retention rate of living was 86.9% when sucrose concentration was 3g/L, was not add 1.34 times of stablizer.Sorbyl alcohol is in the water bath processing process, and change has taken place physicochemical property, and restraining effect appears in work to enzyme.Lactose and NaCl are not obvious to the acid urease provide protection.
B, stablizer are to the effect of immobilization acid urease enzyme membrane:
(1) gets 0.8g chitosan and 0.8g gelatin and be dissolved in the acetate of 100mL mass concentration 1%, 2 mL acid ureases are dissolved in wherein, in mixed solution, add the sucrose of 3g/L or 10% glycerine, place on the magnetic stirring apparatus, after stirring evenly deaeration, double gauze filters, with 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;
The film that makes is cut into 5 cm * 5 cm square of uniform size, is immersed in the glutaraldehyde solution of mass concentration 0.02%, place magnetic agitation 2h on the magnetic stirring apparatus, deionized water wash is removed free glutaraldehyde, drains, the gained fixed film places room temperature preservation, and the results of regular determination enzyme is lived.
The immobilization acid urease enzyme membrane enzyme that adds stablizer is lived: unit membrane area enzyme is lived and is improved 2.62 times, 2.27 times respectively, and glycerine, sucrose are mixed as behind the stablizer, and the work of unit surface enzyme does not have large increase.
Immobilization acid urease enzyme membrane is put in the citrate buffer solution; room temperature preservation was measured the enzyme of immobilization acid urease enzyme membrane and is lived every 20 days, do not add the enzyme loss 34.6% alive of stablizer behind the 60d; with sucrose is the enzyme loss 21.8% alive of stablizer, and glycerol adding is the loss 22.4% alive of protectant enzyme.
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.
(2) in 100 mL system film mixed solution, add 1mL, 1.5mL, 2mL, 2.5mL, 3mL, 3.5mL, 4mL acid urease respectively, behind method system film in the step (1), investigate enzyme rate of recovery influence alive when adding stablizer and not adding stablizer.
Measure as can be known, the suitableeest enzyme concentration of immobilization acid urease enzyme membrane that does not add stablizer is that the 100mL mixed solution adds 2mL enzyme liquid, the enzyme this moment rate of recovery alive is 66.4%, the glycerine of adding 10% is as stablizer, the unit surface enzyme is lived and is improved, and the enzyme rate of recovery alive reaches 82.25%, has improved the utilization ratio of enzyme greatly.
(3) the immobilization acid urease enzyme membrane with 5 cm * 5 cm sizes is put in the 50mL yellow rice wine wine sample 100r/min shaking table, 37 ℃ of temperature, behind the effect 24h, measure its urea concentration, calculate the urea clearance, and the wine sample of getting after the processing is measured local flavor substances content, polyphenol content.
The flavour substances measuring method: the HPLC-GC method is measured
Polyphenol is measured: the Folin-Ciocalteu development process
Measure as can be known, urea content reduces to 10.2 by 32 before and after handling, and the urea clearance reaches 68.3%, and polyphenol content is 0.348mg/mL before handling, and is 0.346 mg/mL after the processing, kind that flavour substances is total and components unchanged.
(4) the immobilization acid urease enzyme membrane of 5 cm * 5 cm sizes is acted on respectively (be made into pH4.5 with citrate buffer solution, urea concentration is 25mgL in 30mL yellow rice wine solution and the 30mL simulation wine
-1, the ethanol volume fraction is 16% simulation wine sample), 37 ℃ of following 100r/min shaking table reaction 12h are one batch, shift out reaction solution, sampling and measuring enzyme activity and urea clearance.Again add fresh solution, survey immobilized enzyme vigor and urea clearance again, so repeatable operation and measure the operational stability of immobilization acid urease enzyme membrane.
Urea clearance: Diacetylmonoxime method
The result shows, in simulation wine sample, react 13 batches after enzyme activity be initial 53%.In the yellow rice wine sample, operational stability decreases, and reuses 7 enzymes and lives and be initial 52%.And under the equal conditions, in simulation wine, preceding four batches of clearances can reach 100%, still can reach 67% after reacting 10 batches, but in yellow rice wine, because yellow rice wine composition more complicated, so after using certain batch continuously, enzyme is lived and descended greatly, the urea clearance also only has 54.9%.
Beneficial effect of the present invention: the active function of enzyme is decided by the structure picture of the complete sum strictness of its molecular structure, at drying and forming-film, freezing the space structure that can cause enzyme in molten grade or the refrigerated storage process repeatedly destroys, thereby lose its original biological activity, it is the key factor of restriction immobilized enzyme widespread use that enzyme loses vigor in immobilized process, adding sucrose or glycerine are stablizer in immobilization acid urease enzyme membrane, can improve the stability of film, and this method does not influence the application of immobilization acid urease enzyme membrane, do not change the character and the state of enzyme membrane, have a good application prospect.
Description of drawings
Fig. 1 adds the immobilization acid urease enzyme membrane preparation technology synoptic diagram of stablizer.
The graph of a relation of Fig. 2 repeatable operation number of times and urea clearance.
The graph of a relation of Fig. 3 repeatable operation and immobilized enzyme vigor.
Embodiment
Embodiment 1:
Embodiment 2:
Get 0.8g chitosan and 0.8g gelatin and be dissolved in the acetate of 100mL mass concentration 1%, 2 mL acid ureases are dissolved in wherein, in mixed solution, add 10% glycerine, place on the magnetic stirring apparatus, stir evenly deaeration after, double gauze filters, with 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; The film that makes is cut into 5 cm * 5 cm square of uniform size, 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, remove free glutaraldehyde (ultraviolet spectrophotometry detection), drain, get immobilization acid urease enzyme membrane.Film is put into the yellow rice wine of 50mL, and 37 ℃ of following 100r/min stirring action 24h measure its urea concentration, calculate the urea clearance, and the wine sample of getting after the processing is measured local flavor substances content, polyphenol content.
| Sample | Yellow rice wine before handling | Yellow rice wine after the processing | Change |
| Urea content (mg/L) | 32 | 13.2 | The 58.8%(clearance) |
| Polyphenol content (mg/mL) | 0.348 | 0.346 | 0.002 |
Yellow rice wine before handling: contain 51 kinds of flavour substancess, 9 kinds of acids wherein, 11 kinds of alcohols, 7 kinds of aldehydes, 24 kinds of ester classes; Yellow rice wine after the processing: contain 50 kinds of flavour substancess, 9 kinds of acids wherein, 11 kinds of alcohols, 8 kinds of aldehydes, 22 kinds of ester classes.Can draw thus, after immobilization is handled, the flavour substances of yellow rice wine not had much affect.
Embodiment 3:
A certain amount of immobilization acid urease enzyme membrane is acted on 30mL yellow rice wine solution and 30mL simulation wine respectively, and (be made into pH4.5 with citrate buffer solution, urea concentration is 25mgL
-1, the ethanol volume fraction is 16% simulation wine sample) in, 37 ℃ of following 100r/min shaking table reaction 12h are one batch, shift out reaction solution, sampling and measuring enzyme activity and urea clearance.Again add fresh solution, survey immobilized enzyme vigor and urea clearance again, like this repeatable operation and measure the operational stability of immobilization acid urease enzyme membrane, as shown in Figures 2 and 3.
The result shows, in simulation wine solution, react 13 batches after enzyme activity be 53%.In the yellow rice wine sample, operational stability decreases, and reuses 7 enzyme work and only be initial 52%.And under the equal conditions, in simulation wine, preceding four batches of urea clearances can reach 100%, still can reach 67% after reacting 10 batches, but in yellow rice wine, because yellow rice wine composition more complicated, so after using certain batch continuously, enzyme fall alive is bigger, the urea clearance is 54.9%.
Claims (4)
1. the method for being maintained fixed an acid urease enzyme membrane stability, it is characterized in that getting 0.8g chitosan and 0.8g gelatin is dissolved in the acetate of 100mL mass concentration 1%, with 2 mL acid ureases be dissolved in wherein the acid urease mixed solution, in described acid urease mixed solution, add final concentration and be the sucrose of 3g/L or final concentration and be 10% glycerine as stablizer, place on the magnetic stirring apparatus, after stirring evenly deaeration, double gauze filters, with 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; The film that makes is cut into 5 cm * 5 cm square of uniform size, place mass concentration 0.02% glutaraldehyde solution, place magnetic agitation 2h on the magnetic stirring apparatus, cover with opaque container, in case glutaraldehyde is seen phototropic, deionized water wash is removed free glutaraldehyde, drain, the immobilization acid urease enzyme membrane of the stability that is maintained, gained immobilization acid urease enzyme membrane places room temperature preservation, and the results of regular determination enzyme is lived.
2. according to the method for described the being maintained fixed acid urease of claim 1 enzyme membrane stability, it is characterized in that described stablizer, all with the citrate buffer solution preparation of pH5.0,4 ℃ store for future use the storage liquid of its stablizer.
3. according to the method for described the being maintained fixed acid urease of claim 1 enzyme membrane stability, it is characterized in that adding acid urease mixed solution behind the stablizer after handling 3h under 70 ℃ of water-baths, with the frozen water cooling, this mixed solution of getting certain volume dilutes 10 times immediately, is used to survey its enzyme and lives.
4. according to the method for described the being maintained fixed acid urease of claim 1 enzyme membrane stability, it is characterized in that the immobilization acid urease enzyme membrane that will add stablizer acts on respectively in 30mL yellow rice wine wine sample or the 30mL simulation wine wine sample, 37 ℃ of following 100r/min shaking table reaction 12h are one batch, shift out reaction solution, sampling and measuring enzyme activity, urea clearance, polyphenol content; Again add fresh described wine sample solution, survey immobilized enzyme vigor and urea clearance again, so repeatable operation and measure the operational stability of immobilization acid urease enzyme membrane.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105462997A (en) * | 2015-12-23 | 2016-04-06 | 江南大学 | New urease prosthetic group gene and application thereof |
| CN112513264A (en) * | 2018-07-31 | 2021-03-16 | 费森尤斯医疗保健控股公司 | Urease purification and purified urease products and sorbent cartridges, systems and methods of use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1036405A (en) * | 1987-07-09 | 1989-10-18 | 武田药品工业株式会社 | Acid urease and production thereof |
| CN101705221A (en) * | 2009-11-23 | 2010-05-12 | 江南大学 | Preparation method and application of immobilized acid urease for wine |
-
2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1036405A (en) * | 1987-07-09 | 1989-10-18 | 武田药品工业株式会社 | Acid urease and production thereof |
| 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 (1)
| Title |
|---|
| 赵圣国等: "脲酶在生物工程中的应用", 《生物技术通报》, no. 3, 31 March 2009 (2009-03-31) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105462997A (en) * | 2015-12-23 | 2016-04-06 | 江南大学 | New urease prosthetic group gene and application thereof |
| CN112513264A (en) * | 2018-07-31 | 2021-03-16 | 费森尤斯医疗保健控股公司 | Urease purification and purified urease products and sorbent cartridges, systems and methods of use thereof |
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| CN102242109B (en) | 2012-11-07 |
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