CN102703419A - Cellulose immobilized cis epoxy succinic acid hydrolase and method for preparing tartaric acid - Google Patents
Cellulose immobilized cis epoxy succinic acid hydrolase and method for preparing tartaric acid Download PDFInfo
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- CN102703419A CN102703419A CN2012101562721A CN201210156272A CN102703419A CN 102703419 A CN102703419 A CN 102703419A CN 2012101562721 A CN2012101562721 A CN 2012101562721A CN 201210156272 A CN201210156272 A CN 201210156272A CN 102703419 A CN102703419 A CN 102703419A
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Abstract
The invention provides a method of cellulose immobilized cis epoxy succinic acid hydrolase. The method comprises the following steps: fusing gene coding cis epoxy succinic acid hydrolase and carbohydrate binding module CMB30, efficiently expressing the fused gene in Escherichia coli, and then carrying out purification and immobilization on fused enzyme in crude enzyme liquid by adopting the cellulose. The invention also provides a method for catalyzing and hydrolyzing epoxy succinic acid to convert into L-tartaric acid or D-tartaric acid by adopting the cellulose immobilized cis epoxy succinic acid hydrolase as biocatalyst. The method has the beneficial effects that: not only is the stability of enzyme obviously improved, but also the cellulose used as purified and fixed substrate is low in price and easy to get by adopting the cellulose immobilized cis epoxy succinic acid hydrolase, and has excellent economic prospect. The method for preparing tartaric acid by adopting the cellulose immobilized cis epoxy succinic acid hydrolase has the advantages of reaction specificity, high product purity, high reaction efficiency and the like.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of method of immobilized cis Epoxysuccinic acid lytic enzyme, and utilize immobilized cis Epoxysuccinic acid enzyme to produce tartaric method.
Background technology
L type or D type tartrate are important chiral separation agent and industrial chemicals.Wherein L (+)-tartrate has good organoleptics property, can add in jam, candy, beverage and the wine with Hydrocerol A, oxysuccinic acid etc., and be important foodstuff additive.L type or D type tartrate utilize also useful as drug industrial raw material of its optical characteristics, as important chiral ligand, can be used for preparing many important chiral catalysts in the organic synthesis in modern times, and are used for the complicated product molecule of composite structure as chiral source.In addition, tartrate also has extensive use in industries such as makeup, plating, process hides, weavings.Therefore the increasing demand of paratartaric acid increases.
Produce L type or D type tartrate at present and mainly contain following several method: extracted form natural plant method, chemical resolution method, microbial transformation glucose fermentation method and enzyme process.Enzyme process is meant with cheap cis-form epoxy succinic acid as producing tartaric raw material, at cis-Epoxysuccinic acid hydratase (L type or D type) or have under the biomass cells catalysis of this enzyme, makes raw material be hydrolyzed to L type or D type tartrate.Prepare tartrate based on enzyme process and possess advantages such as transformation efficiency height, specificity is strong, security is good, its industrial applications is worth the highest.But the stability of enzyme is not good, and the enzyme of the purifying that exsomatizes is inactivation very easily, and therefore, the major applications of cis-Epoxysuccinic acid hydratase all is to adopt intact cell to carry out catalysis at present.But the bio-transformation that utilizes intact cell is the complex process that plurality of enzymes system acts on simultaneously in the whole cell, follows the generation of multiple by product in the fermenting process, can have influence on the separation and purification and the final yield of reacting final product.Advantage of utilizing single-minded High-efficient Production L type of immobilized cis-Epoxysuccinic acid hydratase or D type tartrate to have separately to can not be substituted and potentiality to be exploited greatly.
Summary of the invention
To the problems referred to above, the technical problem that the present invention will solve provides a kind of method of immobilized cis Epoxysuccinic acid lytic enzyme, and utilizes immobilized cis Epoxysuccinic acid lytic enzyme to produce tartaric method.
Technical scheme of the present invention is: the method for cellulose fixed cis-Epoxysuccinic acid hydratase may further comprise the steps:
(1) N end or the C end at coding cis-Epoxysuccinic acid hydratase gene merges glucide binding modules CBM30, obtains fusion expression vector;
(2) fusion expression vector is transformed in the intestinal bacteria, the highly-soluble expressed fusion protein obtains the target thalline;
(3) adopting pH is 7.4 the phosphate buffered saline buffer washing target thalline outstanding cell of laying equal stress on, and re-suspended cell is carried out fragmentation, the centrifugal crude enzyme liquid that obtains;
(4) crude enzyme liquid is mixed with Mierocrystalline cellulose, the centrifugal deposition that obtains, employing pH is 7.4 phosphate buffered saline buffer drip washing deposition and recentrifuge, the gained deposition is the title product immobilized enzyme.
Preferably, said step (2) may further comprise the steps: a. cell cultures: intestinal bacteria are placed nutrient solution, and under 37 ℃, being cultured to OD600 is 0.8-1.0; B. abduction delivering: adding IPTG is 0.2mM to its concentration, and inducing culture is 8 hours under 25 ℃ the temperature condition; C. centrifugal, collect thalline.
Preferably, the broken target thalline of said step (3) can adopt any one in high pressure cell fragmentation, ultrasonication or the N,O-Diacetylmuramidase fragmentation.
Preferably, the centrifugally operated in the said step (3) is at 4 ℃, with 10, and the centrifugal 20min of the rotating speed of 000rpm.
Preferably, described in the said step (4) in Mierocrystalline cellulose and the crude enzyme liquid mass ratio of enzyme be 1: 0.005-1: 0.02.
Adopt cellulose fixed cis-Epoxysuccinic acid hydratase to prepare tartaric method; May further comprise the steps: cellulose fixed cis-Epoxysuccinic acid hydratase is added in the solution of cis-form epoxy succinic acid; At pH is 5-11; Temperature is to react under 25-70 ℃ the condition, promptly obtains title product.
Preferably, when said cellulose fixed cis-Epoxysuccinic acid hydratase is the D type, also contain the Zn of 0.1mM-10mM in the said reaction system
2+, the gained title product is a D type tartrate.
Preferably, the optimal reaction temperature of preparation L type tartrate reaction is 30-50 ℃, and optimum response pH is 8-10; The optimal reaction temperature of preparation D type tartrate reaction is 30-50 ℃, and optimum response pH is 5-9.
The invention has the beneficial effects as follows: the present invention utilizes Mierocrystalline cellulose as immobilized matrix; Use has special adsorbing glucide binding modules CBM30 as affinity tag to Mierocrystalline cellulose; Cis-Epoxysuccinic acid hydratase is carried out amalgamation and expression and prepares immobilized enzyme, compare, following advantage is arranged: the purity that has improved enzyme in the time of (1) immobilized enzyme with the enzyme of on-fixedization; Improve the performance of enzyme, improved the stability of enzyme; (2) Mierocrystalline cellulose is a kind of safe biomacromolecule, and is cheap, and wide material sources possess huge economic outlook; (3) the reaction system impurity of immobilized enzyme is few, the purifying that helps product with separate, do not exist substrate and product to stride the problem of film conveying efficiency.
Compare with the bio-transformation that utilizes intact cell to carry out, adopt cellulose fixed cis-Epoxysuccinic acid hydratase to prepare tartaric method and have reaction and possess specificity, product purity is high, the reaction efficiency advantages of higher.
Embodiment
1. cellulose fixed L type cis-Epoxysuccinic acid hydratase
(1) N end or the C end at coding cis-Epoxysuccinic acid hydratase gene merges glucide binding modules CBM30, obtains fusion expression vector;
(2) single bacterium colony of picking recombinant plasmid transformed on flat board inserts the nutrient solution contain penbritin, and 37 ℃ are cultured to OD600 and reach 0.8-1.0, add IPTG to final concentration 0.2mM, change 25 ℃ of constant temperature shaking tables then over to and continue inducing culture 8 hours.At 4 ℃, centrifugal 15min under the 8000rpm condition collects thalline with bacterium liquid.
(3) employing 40ml pH is 7.4 phosphate buffered saline buffer washing target thalline; Remove residual culture medium solution; And with the phosphate buffered saline buffer re-suspended cell of 20ml; With re-suspended cell broken wall under 22000psi pressure, the bacterium liquid after the fragmentation is centrifugal 20min under the rotating speed of 4 ℃ temperature condition and 10000rpm, the centrifugal crude enzyme liquid that obtains with the broken appearance of high pressure cell;
(4) taking by weighing the 10g Microcrystalline Cellulose mixes with the crude enzyme liquid that 100mL is diluted to about 10mg/mL; Be placed on 4 ℃ of shaking tables and mix 5min; The centrifugal deposition that obtains, employing pH is 7.4 phosphate buffered saline buffer drip washing deposition, removes foreign protein and unconjugated target protein; Recentrifuge, the gained deposition is the title product immobilized enzyme.
Table 1 immobilization fusion enzyme and protoenzyme are in the comparison of differing temps half-life
2. adopt cellulose fixed cis-Epoxysuccinic acid hydratase to prepare tartrate
Take by weighing the wet immobilized enzyme (containing enzyme 5mg approximately) of 5g, add the 20mM phosphoric acid buffer (pH 9.0) that 100ml contains the 250mM cis-form epoxy succinic acid, reaction 2h obtains L type tartrate under 35 ℃ temperature condition, and transformation efficiency reaches 75%.
Claims (8)
1. the method for cellulose fixed cis-Epoxysuccinic acid hydratase is characterized in that: may further comprise the steps:
(1) N end or the C end at coding cis-Epoxysuccinic acid hydratase gene merges glucide binding modules CBM30, obtains fusion expression vector;
(2) fusion expression vector is transformed in the intestinal bacteria, the highly-soluble expressed fusion protein obtains the target thalline;
(3) adopting pH is 7.4 the phosphate buffered saline buffer washing target thalline outstanding cell of laying equal stress on, and re-suspended cell is carried out fragmentation, the centrifugal crude enzyme liquid that obtains;
(4) crude enzyme liquid is mixed with Mierocrystalline cellulose, the centrifugal deposition that obtains, employing pH is 7.4 phosphate buffered saline buffer drip washing deposition and recentrifuge, the gained deposition is the title product immobilized enzyme.
2. the method for cellulose fixed cis-Epoxysuccinic acid hydratase according to claim 1; It is characterized in that: said step (2) may further comprise the steps: a. cell cultures: intestinal bacteria are placed nutrient solution, and under 37 ℃, being cultured to OD600 is 0.8-1.0; B. abduction delivering: adding IPTG is 0.2mM to its concentration, and inducing culture is 8 hours under 25 ℃ the temperature condition; C. centrifugal, collect thalline.
3. the method for cellulose fixed cis-Epoxysuccinic acid hydratase according to claim 1 is characterized in that: the broken target thalline of said step (3) can adopt that high pressure cell is broken, in ultrasonication or the N,O-Diacetylmuramidase fragmentation any one.
4. the method for cellulose fixed cis-Epoxysuccinic acid hydratase according to claim 1 is characterized in that: centrifugal in the said step (3) can be under the rotating speed of 4 ℃ temperature condition and 10000rpm centrifugal 20min.
5. the method for cellulose fixed cis-Epoxysuccinic acid hydratase according to claim 1 is characterized in that: described in the said step (4) in Mierocrystalline cellulose and the crude enzyme liquid mass ratio of enzyme be 1: 0.005-1: 0.02.
6. adopt cellulose fixed cis-Epoxysuccinic acid hydratase to prepare tartaric method; It is characterized in that: may further comprise the steps: cellulose fixed cis-Epoxysuccinic acid hydratase is added in the solution of cis-form epoxy succinic acid; At pH is 5-11; Temperature is to react under 25-70 ℃ the condition, promptly obtains title product tartrate.
7. the cellulose fixed cis-Epoxysuccinic acid hydratase of employing according to claim 6 prepares tartaric method; It is characterized in that: when said cellulose fixed cis-Epoxysuccinic acid hydratase is the D type, also contain the Zn of 0.1mM-10mM in the said reaction system
2+, the gained title product is a D type tartrate.
8. the cellulose fixed cis-Epoxysuccinic acid hydratase of employing according to claim 7 prepares tartaric method, it is characterized in that: the optimal reaction temperature of preparation L type tartrate reaction is 30-50 ℃, and optimum response pH is 8-10; The optimal reaction temperature of preparation D type tartrate reaction is 30-50 ℃, and optimum response pH is 5-9.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103882000A (en) * | 2014-03-17 | 2014-06-25 | 中国科学院过程工程研究所 | Cis-epoxysuccinate hydrolase immobilization method and immobilized enzyme thereof |
| CN105087676A (en) * | 2014-04-21 | 2015-11-25 | 怀来县长城生物化学工程有限公司 | L-(+)-tartaric acid clean production process based on bipolar membrane electroosmosis technology |
| CN105087677A (en) * | 2014-04-21 | 2015-11-25 | 怀来县长城生物化学工程有限公司 | D-(-)-tartaric acid clean production process based on bipolar membrane electroosmosis technology |
| CN105861463A (en) * | 2016-04-12 | 2016-08-17 | 南京工业大学 | Epoxysuccinate hydrolase, and carrier and application thereof |
| CN106497844A (en) * | 2016-12-12 | 2017-03-15 | 常茂生物化学工程股份有限公司 | One plant is produced the tartaric genetic engineering bacteriums of L and its construction method and application |
| CN109266633A (en) * | 2018-09-14 | 2019-01-25 | 华东理工大学 | Carbohydrate binding module and its application in immobilised enzymes preparation and fusion protein purification |
| CN111100835A (en) * | 2020-01-07 | 2020-05-05 | 中国科学院青岛生物能源与过程研究所 | PET degradation biocatalyst and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103882000A (en) * | 2014-03-17 | 2014-06-25 | 中国科学院过程工程研究所 | Cis-epoxysuccinate hydrolase immobilization method and immobilized enzyme thereof |
| CN105087676A (en) * | 2014-04-21 | 2015-11-25 | 怀来县长城生物化学工程有限公司 | L-(+)-tartaric acid clean production process based on bipolar membrane electroosmosis technology |
| CN105087677A (en) * | 2014-04-21 | 2015-11-25 | 怀来县长城生物化学工程有限公司 | D-(-)-tartaric acid clean production process based on bipolar membrane electroosmosis technology |
| CN105861463A (en) * | 2016-04-12 | 2016-08-17 | 南京工业大学 | Epoxysuccinate hydrolase, and carrier and application thereof |
| CN105861463B (en) * | 2016-04-12 | 2019-08-30 | 南京工业大学 | Epoxysuccinic acid hydrolase and its carrier and application |
| CN106497844A (en) * | 2016-12-12 | 2017-03-15 | 常茂生物化学工程股份有限公司 | One plant is produced the tartaric genetic engineering bacteriums of L and its construction method and application |
| CN106497844B (en) * | 2016-12-12 | 2019-05-21 | 常茂生物化学工程股份有限公司 | One plant of genetic engineering bacterium for producing L-TARTARIC ACID and its construction method and application |
| CN109266633A (en) * | 2018-09-14 | 2019-01-25 | 华东理工大学 | Carbohydrate binding module and its application in immobilised enzymes preparation and fusion protein purification |
| CN111100835A (en) * | 2020-01-07 | 2020-05-05 | 中国科学院青岛生物能源与过程研究所 | PET degradation biocatalyst and application thereof |
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Application publication date: 20121003 |