CN103923900A - Preparation method and application of cross-linked enzyme aggregate of bifunctional enzyme for rice wine - Google Patents
Preparation method and application of cross-linked enzyme aggregate of bifunctional enzyme for rice wine Download PDFInfo
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Abstract
The invention provides a preparation method and application of a cross-linked enzyme aggregate of a bifunctional enzyme for rice wine, belonging to the technical field of enzyme preparations and wine making additives. Free urease crude enzyme liquid with urea and ethyl carbamate (EC) degrading activity is extracted from (providencia sp.) JNB815. Free urease is proved to be the bifunctional enzyme with urease and EC degrading enzyme activity. The bifunctional enzyme is prepared into the cross-linked enzyme aggregate by utilizing the carrier-free immobilization technology. Applied to the rice wine, the cross-linked enzyme aggregate can remove urea and EC in the rice wine simultaneously. The urea removal rate of the cross-linked enzyme aggregate during first use can reach 85.35%, the urea removal rate of the cross-linked enzyme aggregate can still reach 69.30% after the cross-linked enzyme aggregate is used six times, and the highest EC removal rate can reach 46.27%. The volatile flavor substances of the rice wine treated by the cross-linked enzyme aggregate are not obviously changed.
Description
Technical field
The purifying of bifunctional enzyme and preparation and application for cross-linked enzyme aggregate for yellow rice wine, belong to zymin, wine brewing additive technology field.
Background technology
Along with the raising of China's living standards of the people, the consumption of the leavened foods such as inebriant rises increasingly.In the process of fermentative production alcohol beverage, there are many unwanted byproducts, urea is exactly one of them, and urea is under acidic conditions, in high-temperature sterilization, still-process or long-time storage process, easily and ethanol formation urethanum.
Urethanum (EC), is the material of 2A class carinogenicity, and trace is present in most of leavened food and alcoholic beverage.External many countries (Canada, the U.S., Japan, European Union etc.) and organize and urethanum in leavened food and alcoholic beverage has been done to limitation requirement, therefore China the content of urethanum in various variant productions is detected imperative.
Urethanum in China's yellow rice wine more than 90% is to derive from reacting of contained urea and ethanol in wine:.Therefore utilize urase that the urea in alcoholic drink is removed in time, significant for the generation of controlling urethanum in wine.Adopt acid urease for wine processing to decoct the yellow rice wine before wine, can remove most of urea in yellow rice wine, reduce the possibility that generates EC, EC degrading enzyme can directly effectively decompose the EC having produced in finished product yellow rice wine.Studies have reported that enterobacteria Enterobacter sp. R-SYB082 can produce a kind of acid urease isozyme, can realize and remove substrate urea and product EC in yellow rice wine simultaneously.
Cross-linked enzyme aggregate is proposed by Sheldon group of Dutch Delft university.Adopt physical method precipitation zymoprotein, obtain enzyme aggregate, more crosslinked with linking agent, prepare cross-linked enzyme aggregate.Its active and stability can compare favourably with crosslinked enzyme crystal technology, and preparation process does not need crystallization, the purification step of complicated and time consumption, is the novel enzyme process for fixation of a kind of low cost, easy to operate, high activity and stability.There is low to enzyme purity requirement, high without carrier, unit volume activity, space efficiency advantages of higher.The present invention adopts the resolvase extracting in (Providencia sp.) JNB815 to make cross-linked enzyme aggregate, and urea and the urethanum that can degrade in yellow rice wine have a dual effect to the control of EC in yellow rice wine simultaneously.
Summary of the invention
The object of this invention is to provide a kind of yellow rice wine purifying process of bifunctional enzyme and the preparation and application method of cross-linked enzyme aggregate.
Technical scheme of the present invention, the preparation method of bifunctional enzyme for a kind of natural materials compound system immobilization wine, the resolvase that will extract from the broken liquid of (Providencia sp.) JNB815 somatic cells, carry out purifying and SDS-PAGE gel electrophoresis checking through ethanol precipitation, DEAE-FF ion exchange chromatography, Superdex 200 gel chromatographies, preliminary proof is the bifunctional enzyme that same has two kinds of enzymic activitys.Resolvase is crosslinked through ethanol precipitation and genipin, makes cross-linked enzyme aggregate.
1. its technique is:
(1) free urase extracts:
Bacterial classification Providence (Providencia sp.) JNB815; This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number: CGMCC No.8326.
Seed culture: seed culture medium composition is in g/L: glucose 20, peptone 10, extractum carnis 10, yeast extract paste 5, KH2PO4 2, NaCl 5, NaAc 2, urea 5, adjusts pH 7.0 with NaOH, prepares with pure water; 3%, 37 DEG C of shaking table of inoculum size is cultivated 8-12 h, rotating speed 150 rpm;
Fermentation culture: fermention medium composition is in g/L: glucose 20, peptone 10, extractum carnis 5, yeast extract paste 5, KH2PO4 2, NaCl 5, NaAc 2, urea 5, four water manganous sulfates 0.05, nickel sulfate hexahydrate 0.05, HCl adjusts pH5.5, prepares with pure water; 5%, 37 DEG C of shaking table of inoculum size is cultivated 20-24 h, rotating speed 150 rpm;
Above bacterial classification is after seed culture, fermentation culture, gained fermented liquid is after 4 DEG C, centrifugal 5 min of 8000rpm, abandoning supernatant, gained thalline deionized water washed twice, wash once with the citric acid-sodium citrate damping fluid of pH4.5,50mmol/L again, after the centrifugal 5min of 8000r/min, obtain full cell; The full cell obtaining is diluted to 1/10 of original fermented solution volume with citric acid-sodium citrate damping fluid, and Eddy diffusion thalline, stirs evenly, and carries out ultrasonic disruption; Ultrasonic disruption condition is 300W, 15min, broken time 3s, interval time 3s, treatment capacity 35mL; Broken liquid, at 4 DEG C, the centrifugal 20min of 10000rpm, is collected supernatant liquor, obtains free urase crude enzyme liquid;
(2) purifying of free urase:
A, ethanol precipitation: by step (1) by (
providenciasp.) the free urase crude enzyme liquid that JNB815 extracts, adopting successively mass concentration is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% ethanol precipitation, the centrifugal 20min of 10000r/min, gained precipitation is redissolved in pH6.8,25mmolL
-1in sodium phosphate buffer, measure the enzyme of supernatant liquor and live and draw alcohol precipitation curve;
B, DEAE-FF ion exchange chromatography: DEAE-FF chromatography column is in advance with pH6.8,25mmolL
-1sodium phosphate buffer balance is complete, and the enzyme liquid loading that 5mL alcohol precipitation is obtained, with 0～1.0 molL of described sodium phosphate buffer preparation
-1naCl solution carry out gradient elution, flow velocity 1.0 mLmin
-1, collect protein peak, measure each peak urase and EC degradation enzyme activity, be associated with enzyme part alive, 4 DEG C save backup;
C, Superdex 200 gel permeation chromatographies: with pH6.8,25mmolL
-12～3 column volumes of sodium phosphate buffer balance Superdex 200 chromatography column, after complete equipilibrium, the enzyme liquid that 1mL step b is collected is added on chromatography column, carry out wash-out with sodium phosphate buffer, collect, be associated with enzyme part alive, ultrafiltration and concentration obtains the free urase after purifying, and 4 DEG C save backup;
(3) preparation of crosslinking enzyme aggressiveness: the ethanol that the free urase of step (2) c gained is 10% by mass concentration is removed after impurity, adopt the ethanol that final concentration is 60% to precipitate, gained precipitation is redissolved in 5ml in the citric acid-sodium citrate damping fluid containing pH4.5, the 50mmol/L of 60% ethanol, add the 0.3%(m/v of reaction system cumulative volume) genipin (Genipin, molecular formula C
11h
14o
5), under room temperature, being cross-linked 2.5h, mixing solutions is placed in 4 DEG C of refrigerators and generates cross-linked enzyme aggregate CLEAs; At 10000 rmin
-1after centrifugal 20min, abandon supernatant, precipitation is used ultrapure water washed twice, and centrifugal acquisition cross-linked enzyme aggregate CLEAs stores in 4 DEG C of refrigerators.
2. the character of purifying enzyme: after the ethanol impurity elimination of employing 10%, the precipitation of collection ethanol final concentration between 10%-60% has the activity of urase and urethanum degrading enzyme.When DEAE-FF ion exchange chromatography, at 0.35 molL
-1, 0.55 molL
-1time an each elution peak, wash-out concentration 0.35 molL of occurring
-1time the elution peak that occurs there is urase and EC enzyme enzyme and live.When Superdex 200 gel chromatography, be that a large peak appears in 7.2 mL left and right at elution volume, there are these two kinds of activity of urase and urethanum degrading enzyme.The void volume of corresponding Superdex 200 gel columns of this elution volume.In the process of above three step purifying, the Peak Activity of two kinds of enzymes is overlapping, and molecular weight is consistent.Can be found out by SDS-PAGE electrophoresis, through ethanol precipitation, separating effect is comparatively obvious, has removed partial impurities; DEAE-FF has removed two or three kind of foreign protein after separating, and each band boundary is clear, can see some main bands; Separate via Superdex 200 the Peak Activity purification finally obtaining higher, object band number is 2, and supposition may be the subunit of two kinds of different sizes of the same enzyme.
Resolvase enzyme activity determination method: adopt indophenol blue reaction (Berthelot reaction) colorimetry.
Enzyme activity definition: at normal pressure, 37 DEG C, under pH4.5 condition, it is an enzyme activity unit that per minute decomposition substrate urea or urethanum produce 1 μ mol ammonia.Wherein, to adopt respectively urea and EC be substrate to the mensuration of urea degrading enzymatic and EC degrading enzyme vigor.
Cross-linked enzyme aggregate enzyme activity determination method: get in 3% the urea or EC solution of pH4.5 that 0.1 g cross-linked enzyme aggregate immerses respectively the citric acid-sodium citrate damping fluid preparation of certain volume, be incubated after 20 min 10000 rmin in 37 DEG C of constant water bath box
-1its supernatant of centrifuging and taking, remaining determination step is identical with resolvase vitality test step.Enzyme activity defines same resolvase.
The mensuration of protein content: adopt coomassie brilliant blue staining method, using bovine serum albumin as standard protein.
The measuring method of urea content: diacetyl monoxime method.
The measuring method of EC content: adopt combined gas chromatography mass spectrometry.
The mensuration of Flavor in Rice Wine: adopt head space-solid-phase microextraction technology.
3, the application of bifunctional enzyme cross-linked enzyme aggregate for the yellow rice wine preparing: get commercially available yellow rice wine solution, be loaded in reaction vessel, add 25 mgL
-1cross-linked enzyme aggregate, every reaction 6 h are one batch, with 10000 rmin
-1centrifugal 20 min reclaim CLEAs, and add new lot yellow rice wine of the same race to process, and repeatedly, measure each reclaimed CLEAs quality and the urea clearance of yellow rice wine, react 6 batches after CLEAs weight in wet base only reduce by 0.18 g, there is no considerable change; The stability that cross-linked enzyme aggregate is described is better, and leakage or the dissolution phenomena of enzyme in use do not occur.
To the clearance of urea, first use can reach 85.35% to described cross-linked enzyme aggregate, use 6 times after urea clearance still can reach 69.30%; Clearance to EC is up to 46.27%, and reusability is better; Through cross-linked enzyme aggregate yellow rice wine after treatment, its volatile flavor substance is without considerable change.
Beneficial effect of the present invention: this bifunctional enzyme is made after cross-linked enzyme aggregate can be stablized and play a role in wine.Add cross-linked enzyme aggregate in yellow rice wine after, to the clearance of urea, first use can reach 85.35%, use 6 times after urea clearance still can reach 69.30%.Clearance to EC reaches as high as 46.27%, and cross-linked enzyme aggregate be convenient to reclaim, 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 cross-linked enzyme aggregate, just can reduce urea and EC content.And with respect to other process for fixation, cost is lower, it is a kind of method of removing urea and EC in wine comparatively desirable time.
Biological material specimens preservation: the bacterial strain of acid urease and EC degrading enzyme is produced in a strain, Classification And Nomenclature is Providence (Providencia sp.) JNB815, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, be called for short CGMCC, address: No. 3 Institute of Microorganism, Academia Sinica of BeiChen West Road, Chaoyang District, BeiJing City l institute, its deposit number is: CGMCC No.8326, preservation date is on October 12nd, 2013.
Brief description of the drawings
Fig. 1 alcohol precipitation curve.
The DEAE-FF ion exchange chromatography collection of illustrative plates of Fig. 2 urase.
The Superdex 200 gel chromatography collection of illustrative plates of Fig. 3 urase.
Fig. 4 SDS-PAGE electrophorogram.1, Marker; 2, thick enzyme; 3, alcohol precipitation; 4, DEAE; 5, Superdex 200.
Fig. 5 CLEAs is applied to the process flow sheet in membrane reactor.
The volatile flavor substance collection of illustrative plates of Fig. 6 yellow rice wine.(a figure represents former yellow rice wine, and b figure represents cross-linked enzyme aggregate yellow rice wine after treatment).
Embodiment
Embodiment 1
Resolvase extracts: deposit number is: the Providence of CGMCC No.8326 (
providenciasp.) JNB815 bacterial classification obtains a certain amount of thalline through seed culture and fermentation culture, by after 4 DEG C of fermented liquids, centrifugal 5 min of 8000rpm, abandon supernatant, gained thalline deionized water washed twice, the citric acid-sodium citrate damping fluid of pH4.5,50mmol/L washs once, the full cell of centrifugal acquisition; By obtain complete for cell the citric acid-sodium citrate damping fluid of pH4.5,50mmol/L be diluted to 1/10 of original fermented solution volume, Eddy diffusion thalline, stir evenly, carry out ultrasonic disruption, ultrasonic disruption condition is 300 W, 15 min, broken times 3 s, interval times 3 s, treatment capacity 35 mL; Broken liquid, at 4 DEG C, centrifugal 20 min of 10000 rpm, is collected supernatant liquor, is free urase crude enzyme liquid;
Will (
providenciasp.) the free urase that JNB815 extracts, through ethanol precipitation, DEAE-FF ion exchange chromatography, Superdex 200 gel permeation chromatographies.The alcohol precipitation grading range of urine enzyme and urethanum degrading enzyme is identical, and Peak Activity is overlapping.It is same bifunctional enzyme that preliminary proof has these two kinds of active enzymes.Concrete steps are:
Ethanol precipitation: will by (
providenciasp.) the free urase that JNB815 extracts, adopts 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% ethanol precipitation successively, centrifugal, and gained precipitation is redissolved in pH6.8,25mmolL
-1in sodium phosphate buffer, measure the enzyme of supernatant liquor and live and draw alcohol precipitation curve, curve as shown in Figure 1.
DEAE-FF ion exchange chromatography: DEAE-FF chromatography column is in advance with 25 mmolL
-1, pH6.8 sodium phosphate buffer balance is complete, the enzyme liquid loading that 5 mL alcohol precipitations are obtained, with 0～1.0 molL
-1naCl(sodium phosphate level pad preparation) solution carries out gradient elution, flow velocity 1.0 mLmin
-1, collect protein peak, measure each peak enzyme and live, be associated with enzyme part alive, 4 DEG C save backup, and the DEAE-FF ion exchange chromatography collection of illustrative plates of urase is as shown in Figure 2.
Superdex 200 gel permeation chromatographies: with 25 mmolL
-12～3 column volumes of sodium phosphate buffer (pH6.8) balance Superdex 200 chromatography column, after complete equipilibrium, the enzyme liquid of 1 mL DEAE-FF ion exchange chromatography collection step is added on chromatography column, carry out wash-out with sodium phosphate level pad, collect, be associated with enzyme part alive, after ultrafiltration and concentration, 4 DEG C save backup.The Superdex 200 gel chromatography collection of illustrative plates of urase as shown in Figure 3.(
providenciasp.) purification result of JNB815 product enzyme is as shown in table 1.
Table 1
providenciasp. JNB815 produces the purification result of enzyme
After the ethanol impurity elimination of employing 10%, the precipitation of collection ethanol final concentration between 10%-60% has the activity of urase and urethanum degrading enzyme.When DEAE-FF ion exchange chromatography, at 0.35 molL
-1, 0.55 molL
-1time an each elution peak, wash-out concentration 0.35 molL of occurring
-1time the elution peak that occurs there is urase and EC enzyme enzyme and live.When Superdex 200 gel chromatography, be that a large peak appears in 7.2 mL left and right at elution volume, there are these two kinds of activity of urase and urethanum degrading enzyme.The void volume of corresponding Superdex 200 gel columns of this elution volume.In the process of above three step purifying, the Peak Activity of two kinds of enzymes is overlapping, and molecular weight is consistent.By Fig. 4, SDS-PAGE electrophorogram can be found out, process ethanol precipitation, and separating effect is comparatively obvious, has removed partial impurities; DEAE FF has removed two or three kind of foreign protein after separating, and each band boundary is clear, can see some main bands; Separate via Superdex 200 the Peak Activity purification finally obtaining higher, object band number is 2.
Embodiment 2
Free urase is removed after impurity with 10% ethanol, adopt the ethanol that final concentration is 60% to carry out fractionation precipitation, gained precipitation is redissolved in the citric acid-sodium citrate damping fluid of a small amount of pH4.5,50mmol/L containing 60% ethanol, add the 0.3%(m/v of reaction system cumulative volume) genipin (Genipin, molecular formula C
11h
14o
5) be cross-linked under room temperature, reaction 2.5h, mixing solutions is placed in 4 DEG C of refrigerators and generates CLEAs.10000 rmin
-1the centrifugal supernatant of abandoning, precipitation is used ultrapure water washed twice, and centrifugal acquisition cross-linked enzyme aggregate is stored in 4 DEG C of refrigerators.
Embodiment 3
In commercially available yellow rice wine, add 25 mgL
-1urea, is mixed with certain density yellow rice wine sample solution, adds the cross-linked enzyme aggregate of embodiment 2 gained to process.Container is placed in magnetic stirring apparatus, and every reaction 6 h are one batch, 10000 rmin
-1centrifugal 20 min reclaim CLEAs, and add new lot yellow rice wine of the same race to process, and repeatedly, measure each reclaimed CLEAs quality and the urea clearance of yellow rice wine.After reacting 6 batches, CLEAs weight in wet base only reduces by 0.18 g, there is no considerable change.The stability that cross-linked urease congeries is described is better, and leakage or the dissolution phenomena of enzyme in use do not occur.The urea clearance of the 1st batch and the 6th batch is respectively 85.35% and 69.30%, and reusability is better.
CLEAs is applied to process flow sheet in membrane reactor as shown in Figure 5.
The reusability of table 2 cross-linked urease congeries in yellow rice wine
Embodiment 4
As shown in Figure 5, by the cross-linked enzyme aggregate of embodiment 2 gained, add respectively difference to measure in reaction vessel, add the yellow rice wine of same volume to process.Stir with certain rotating speed, process after yellow rice wine 12 h, reacted yellow rice wine sample separates through microfiltration membrane tripping device.The variation of EC concentration, urea concentration and volatile flavor substance before and after assaying reaction respectively.As shown in Table 3 and Table 4, along with the increase of cross-linked urease congeries add-on, urea clearance and the EC clearance of yellow rice wine increase.When urase enzyme concentration is 0.15 UmL
-1time, urea clearance reaches 80.49%.When EC enzyme enzyme concentration is 0.17 UmL
-1time, EC clearance is 46.27%.From GC-MS atlas analysis result, through cross-linked enzyme aggregate yellow rice wine after treatment, the contamination of its volatile flavor substance changes not obvious.The volatile flavor substance collection of illustrative plates of yellow rice wine as shown in Figure 6.
The urea clearance of table 3 CLEAs to yellow rice wine sample
The EC clearance of table 4 CLEAs to yellow rice wine sample
Claims (4)
1. a preparation method for bifunctional enzyme cross-linked enzyme aggregate for yellow rice wine, is characterized in that step is:
(1) free urase extracts:
Bacterial classification Providence (
providenciasp.) JNB815; This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number: CGMCC No.8326;
Seed culture: seed culture medium forms in g/L: glucose 20, peptone 10, extractum carnis 10, yeast extract paste 5, KH
2pO
42, NaCl 5, NaAc 2, urea 5, adjusts pH 7.0 with NaOH, prepares with pure water; 3%, 37 DEG C of shaking table of inoculum size is cultivated 8-12 h, rotating speed 150 rpm;
Fermentation culture: fermention medium forms in g/L: glucose 20, peptone 10, extractum carnis 5, yeast extract paste 5, KH
2pO
42, NaCl 5, NaAc 2, urea 5, four water manganous sulfates 0.05, nickel sulfate hexahydrate 0.05, HCl adjusts pH5.5, prepares with pure water; 5%, 37 DEG C of shaking table of inoculum size is cultivated 20-24 h, rotating speed 150 rpm;
Above bacterial classification is after seed culture, fermentation culture, gained fermented liquid is after 4 DEG C, centrifugal 5 min of 8000rpm, abandoning supernatant, gained thalline deionized water washed twice, wash once with the citric acid-sodium citrate damping fluid of pH4.5,50mmol/L again, after the centrifugal 5min of 8000r/min, obtain full cell; The full cell obtaining is diluted to 1/10 of original fermented solution volume with citric acid-sodium citrate damping fluid, and Eddy diffusion thalline, stirs evenly, and carries out ultrasonic disruption; Ultrasonic disruption condition is 300W, 15min, broken time 3s, interval time 3s, treatment capacity 35mL; Broken liquid, at 4 DEG C, the centrifugal 20min of 10000rpm, is collected supernatant liquor, obtains free urase crude enzyme liquid;
(2) purifying of free urase:
A, ethanol precipitation: by step (1) by (
providenciasp.) the free urase crude enzyme liquid that JNB815 extracts, adopting successively mass concentration is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% ethanol precipitation, the centrifugal 20min of 10000r/min, gained precipitation is redissolved in pH6.8,25mmolL
-1in sodium phosphate buffer, measure the enzyme of supernatant liquor and live and draw alcohol precipitation curve;
B, DEAE-FF ion exchange chromatography: DEAE-FF chromatography column is in advance with pH6.8,25mmolL
-1sodium phosphate buffer balance is complete, and the enzyme liquid loading that 5mL alcohol precipitation is obtained, with 0～1.0 molL of described sodium phosphate buffer preparation
-1naCl solution carry out gradient elution, flow velocity 1.0 mLmin
-1, collect protein peak, measure each peak urase and EC degradation enzyme activity, be associated with enzyme part alive, 4 DEG C save backup;
C, Superdex 200 gel permeation chromatographies: with pH6.8,25mmolL
-12～3 column volumes of sodium phosphate buffer balance Superdex 200 chromatography column, after complete equipilibrium, the enzyme liquid that 1mL step b is collected is added on chromatography column, carry out wash-out with sodium phosphate buffer, collect, be associated with enzyme part alive, ultrafiltration and concentration obtains the free urase after purifying, and 4 DEG C save backup;
(3) preparation of cross-linked enzyme aggregate: the ethanol that the free urase of step (2) c gained is 10% by mass concentration is removed after impurity, adopt the ethanol that final concentration is 60% to precipitate, gained precipitation is redissolved in 5mL in the citric acid-sodium citrate damping fluid containing pH4.5, the 50mmol/L of 60% ethanol, add 0.3% of reaction system cumulative volume, m/v, genipin, under room temperature, be cross-linked 2.5h, mixing solutions is placed in 4 DEG C of refrigerators and generates cross-linked enzyme aggregate CLEAs; At 10000 rmin
-1after centrifugal 20min, abandon supernatant, precipitation is used ultrapure water washed twice, and centrifugal acquisition cross-linked enzyme aggregate CLEAs stores in 4 DEG C of refrigerators.
2. the preparation method of bifunctional enzyme cross-linked enzyme aggregate for yellow rice wine according to claim 1, is characterized in that the purifying of free urase:
A, ethanol precipitation: ethanol final concentration is between 10%-60% time, and the precipitation of collection has the activity of urase and urethanum degrading enzyme;
When b, DEAE-FF ion exchange chromatography, at 0.35 molL
-1, 0.55 molL
-1time an each elution peak, wash-out concentration 0.35 molL of occurring
-1time the elution peak that occurs there is urase and EC enzyme enzyme and live;
When c, Superdex 200 gel chromatography, in the time that elution volume is 7.2 mL, there is a large peak, there are these two kinds of activity of urase and urethanum degrading enzyme.
3. the application of bifunctional enzyme cross-linked enzyme aggregate for the yellow rice wine preparing by method described in claim 1, is characterized in that: get commercially available yellow rice wine solution, be loaded in reaction vessel, add 25 mgL
-1cross-linked enzyme aggregate, every reaction 6 h are one batch, with 10000 rmin
-1centrifugal 20 min reclaim CLEAs, and add new lot yellow rice wine of the same race to process, and repeatedly, measure each reclaimed CLEAs quality and the urea clearance of yellow rice wine, react 6 batches after CLEAs weight in wet base only reduce by 0.18 g, there is no considerable change; The stability that cross-linked enzyme aggregate is described is better, and leakage or the dissolution phenomena of enzyme in use do not occur.
4. the application of bifunctional enzyme cross-linked enzyme aggregate for yellow rice wine according to claim 3, is characterized in that: to the clearance of urea, first use can reach 85.35% to described cross-linked enzyme aggregate, use 6 times after urea clearance still can reach 69.30%; Clearance to EC is up to 46.27%, and reusability is better; Through cross-linked enzyme aggregate yellow rice wine after treatment, its volatile flavor substance is without considerable change.
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| CN105462997A (en) * | 2015-12-23 | 2016-04-06 | 江南大学 | New urease prosthetic group gene and application thereof |
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| CN105969694A (en) * | 2016-06-20 | 2016-09-28 | 江南大学 | Providencia for synthesizing ethanol-resistant urethanase and urease |
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| CN105969694A (en) * | 2016-06-20 | 2016-09-28 | 江南大学 | Providencia for synthesizing ethanol-resistant urethanase and urease |
| CN105950596B (en) * | 2016-06-20 | 2019-06-21 | 江南大学 | A kind of difunctional acid urease gene and its expression and application |
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| CN106282071B (en) * | 2016-10-26 | 2019-07-23 | 江南大学 | The bacillus amyloliquefaciens of one plant of degradation urethanes and urea |
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| CN109735525B (en) * | 2018-12-28 | 2021-05-07 | 浙江工业大学 | Preparation method of cross-linked enzyme polymer |
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