Method for preparing l-2-aminobutyric acid through whole cell biotransformation

An aminobutyric acid and biotransformation technology, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., can solve problems such as many steps, and achieve the effects of efficient conversion, cost reduction, and cost reduction

Active Publication Date: 2018-04-27
上海弗凯生物科技有限公司
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  • Abstract
  • Description
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  • 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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PUM

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Abstract

The invention relates to a method for preparing L-2-aminobutyric acid through biotransformation of whole cells. The method is as follows: expressing threonine deaminase and leucine dehydrogenase in series, cloning the chloramphenicol resistance expression vector, realizing the co-expression of the two enzymes in the same bacterium; cloning the formate enzyme into kalamycin Express on the resistant pET-28a vector, and express in tandem with the pcnB gene in the rate-limiting step of the coenzyme I metabolic pathway in E. coli to achieve high expression of the two enzymes; transform the two resistant expression vectors into the same bacterium Co-expression in the same genus of the four enzymes was achieved, and the expression of four enzymes in the same genus was realized; the whole cell of E. coli containing the four enzymes was fermented and expressed in E. coli, and the synthesis from threonine to L-2-aminobutyric acid was realized. Bioreductive transformation. The invention realizes high-efficiency conversion of 2-ketobutyric acid in the conversion system without adding coenzymes, and has low cost and simple method.

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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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/00C12R1/19
Inventor 郁庆明
Owner 上海弗凯生物科技有限公司
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