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Method for purifying and immobilizing gamma-lactamase and splitting (+/-) gamma-lactam by one step

A technology of lactamase and lactam hydrolase is applied in the field of enzyme engineering to achieve the effects of reducing the amount of enzyme and the loss of enzyme activity, reducing production costs and simplifying the purification process

Inactive Publication Date: 2009-09-30
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] (1) The immobilized enzyme is easily separated from the substrate and product;

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0029] 4. 332 mg of enzyme was immobilized on an affinity column, and the column was incubated in a water bath at 30°C, 2g L -1 Substrate at 0.22mL·min -1 The flow rate is converted through the column, the conversion rate reaches 90%, and the e.e value reaches 99.02%.

example 2

[0031] 4. 332 mg of enzyme was immobilized on an affinity column, and the column was incubated in a water bath at 60°C, 20g L -1 Substrate at 0.22mL·min -1 The flow rate is converted through the column, the conversion rate reaches 88%, and the e.e value reaches 99.1%.

example 3

[0033] 4. 332 mg of enzyme was immobilized on an affinity column, and the column was incubated in a water bath at 60 ° C, 5 g L -1 Substrate at 0.5mL·min -1 The flow rate is converted through the column, the conversion rate reaches 91%, and the e.e value reaches 99%.

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PUM

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Abstract

The invention belongs to the field of enzyme engineering, and particularly relates to a method for purifying and immobilizing gamma-lactamase and splitting (+ / -) gamma-lactam by one step. Gamma-lactam hydrolase is immobilized on a Ni-NTA agarose affinity column, and (+) gamma-lactam is hydrolyzed on the column so as to split the (+ / -) gamma-lactam. Plasmids of heat-resistant gamma-lactam hydrolase genes with sulfolobus solfataricus are transferred into colon bacillus to construct gene engineering bacteria capable of expressing the gamma-lactam hydrolase with histidine marks at an N end and a C end or gene engineering bacteria capable of expressing the gamma-lactam hydrolase with histidine marks at only one end of the N end and the C end. And then, the enzyme with the histidine marks is further purified and immobilized on the Ni-NTA agarose affinity column, and a (+ / -) gamma-lactam substrate is continuously converted on the column under the conditions that the pH value is 5 to 10 and the temperature is between 30 and 100 DEG C; the conversion rate can be kept at more than 80 percent; and the optical purity of a product is more than 99 percent.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, specifically a new technology of immobilized γ-lactam hydrolase to split (±) 2-azabicyclo-[2.2.1]-heptane-5-en-3-one ( γ-lactam). Background technique [0002] (-)γ-lactam ((-)2-azabicyclo-[2.2.1]-heptane-5-en-3-one) is an important intermediate in the synthesis of many biologically active compounds, including abaca Wei and other anti-AIDS chiral drugs. [0003] The methods for preparing optically pure chiral compounds by resolution of chiral compounds can be summarized as follows: (1) according to the starting materials, they are divided into racemic resolution and asymmetric synthesis; (2) according to the means used, they are divided into chemical There are two types of method and biocatalysis. Microbial enzymatic resolution technology is favored by scientific researchers because of its characteristic of enzymatic catalytic reaction. Microbial enzymatic resolution is a method of splitting...

Claims

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Application Information

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IPC IPC(8): C12N11/10C12N11/04C12P13/02
Inventor 郑国钧林建东王建军张星曹燕萍
Owner BEIJING UNIV OF CHEM TECH
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