Cellulose immobilized cis epoxy succinic acid hydrolase and method for preparing tartaric acid

A technology of epoxy succinic acid and cellulose, applied in the field of bioengineering, can solve the problems affecting the separation and purification of the final reaction product and the final yield, poor enzyme stability, easy inactivation of enzymes, etc. , less impurities, the effect of improving the performance of the enzyme

Inactive Publication Date: 2012-10-03
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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Problems solved by technology

However, the stability of the enzyme is not good, and the enzyme purified in vitro is easily inactivated. Therefore, most of the current applications of cis-epoxysuccinate hydrolase are catalyzed by intact cells.
However, the biotransformation using intact cells is a complex process in which multiple enzyme systems act simultaneously in the whole cell. The fermentation process is accompanied by the generation of various by-products, which will affect the separation, purification and final yield of the final product of the reaction.

Method used

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  • Cellulose immobilized cis epoxy succinic acid hydrolase and method for preparing tartaric acid

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Embodiment Construction

[0019] 1. Cellulose-immobilized L-type cis-epoxysuccinate hydrolase

[0020] (1) Fusing the carbohydrate binding module CBM30 at the N-terminal or C-terminal of the gene encoding cis-epoxysuccinate hydrolase to obtain a fusion expression vector;

[0021] (2) Pick a single colony transformed with the recombinant plasmid on the plate, insert it into the culture medium containing ampicillin, cultivate it at 37°C until the OD600 reaches 0.8-1.0, add IPTG to the final concentration of 0.2mM, and then transfer it to a constant temperature shaker at 25°C The induction culture was continued for 8 hours. The bacterial liquid was centrifuged at 4°C and 8000 rpm for 15 minutes to collect the bacterial cells.

[0022] (3) Wash the target cells with 40ml of phosphate buffer with a pH of 7.4, remove the residual medium solution, resuspend the cells with 20ml of phosphate buffer, and break the resuspended cells under a pressure of 22000psi with a high-pressure cell disruptor. wall, the bro...

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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 [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for immobilizing cis-epoxysuccinate hydrolase and a method for producing tartaric acid by using the immobilized cis-epoxysuccinate. Background technique [0002] L-type or D-type tartaric acid is an important chiral resolving agent and chemical raw material. Among them, L(+)-tartaric acid has good sensory properties, and can be added to jam, candy, beverage and wine together with citric acid and malic acid, and is an important food additive. L-type or D-type tartaric acid can also be used as a raw material for pharmaceutical industry by taking advantage of its optical properties. It can be used as an important chiral ligand in modern organic synthesis, and can be used to prepare many important chiral catalysts and used as a chiral source. Synthesis of structurally complex product molecules. In addition, tartaric acid is also widely used in cosmetics, electropl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/12C12P7/46
Inventor 崔球王珊崔古贞冯银刚
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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