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Method for preparing L-2-aminobutyric acid through whole-cell bioconversion

A kind of aminobutyric acid, biotransformation technology

Active Publication Date: 2015-04-22
NANJING Y BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current biotransformation method has many steps, involves enzyme preparation, and needs to add coenzymes to the bioreduction transformation system, and the cost cannot be compared with chemical synthesis.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0013] Example 1: Replace the resistance and origin of replication of the vector of pET28 with the chloramphenicol resistance and origin of replication of pLys S, and then clone threonine deaminase and leucine dehydrogenase into the vector in series ; The formate enzyme and pcnB genes were connected in series to the pET28a vector; two expression vectors with different resistances were simultaneously transferred into Escherichia coli BL21 (DE3) expression host bacteria, and under the induction of IPTG, the simultaneous expression of four enzymes was realized. Express.

[0014] Escherichia coli fermentative expression containing four enzymes was used to achieve bioreductive transformation from threonine to L-2-aminobutyric acid with whole cells.

Embodiment 2

[0016] The bacterial cells expressing the above four enzymes at the same time were prepared into a suspension, added a certain amount of ammonium formate and threonine, and transformed at 30 degrees, and then continuously added threonine until the final concentration was 0.8 M, L- The conversion of 2-aminobutyric acid basically stops, and the bacterial cells are removed by centrifugation. The supernatant is concentrated to dryness, washed with methanol to remove salt, and the purity of 98% of L-2-aminobutyric acid is obtained, and the EE value is above 99%.

[0017] In this application, the threonine deaminase and leucine dehydrogenase are cloned into a chloramphenicol-resistant low-copy expression vector, and the active low-formic acid is cloned into the pET28a vector. In order to increase the expression of the endogenous coenzyme I of the engineering bacteria, the enzyme pcnB gene of the rate-limiting step in the synthesis of the endogenous coenzyme I of Escherichia coli and ...

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Abstract

The invention relates to a method for preparing L-2-aminobutyric acid through whole-cell bioconversion. The method disclosed by the invention comprises the following steps of: carrying out tandem expression on threonine deaminase and leucine dehydrogenase, and cloning on a chlorampenicol resistant expression vector so as to realize co-expression of the two enzymes in the same bacterium; cloning formilase on a kalamycin resistant pET-28a vector to realize expression, and carrying out tandem expression with a pcnB gene in a rate-limiting step of coenzyme I metabolic pathways in escherichia coli so as to realize high expression of the two enzymes; converting the expression vector having two resistances into the same bacterium to carry out co-expression so as to realize expression of four enzymes in the same host bacterium; and carrying out fermentation expression of whole-cell for escherichia coli containing the four enzymes by using escherichia coli so as to realize biological reduction conversion from threonine to L-2-aminobutyric acid. According to the invention, high-efficiency conversion of 2-ketobutyric acid can be realized without addition of coenzyme; the cost is low; and the method is simple.

Description

technical field [0001] The invention relates to a method for preparing L-2-aminobutyric acid through biotransformation of whole cells. Background technique [0002] L-2-aminobutyric acid is an important pharmaceutical intermediate and chemical raw material, and the market demand is very large, with an annual market of more than 2,000 tons. At present, the industry mainly uses chemical methods for synthesis. The advantage of the chemical method is that the preparation cost is low, but it pollutes the environment seriously. With the improvement of the country's requirements for environmental protection, the preparation of L-2-aminobutyric acid by the biological method is favored by people. The advantage of the biological method is that the pollution is small and the environment Friendly, which belongs to green industrial products, is the direction of development. However, the current biotransformation method has many steps, involves enzyme preparation, and needs to add coenz...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12R1/19
Inventor 郁庆明
Owner NANJING Y BIO PHARMA CO LTD
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