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Preparation method of organic alcohol oxidase

An alcohol oxidase and organic technology, applied in the direction of oxidoreductase, introduction of foreign genetic material by using carrier, recombinant DNA technology, etc., can solve problems such as poor thermal stability, achieve convenient transportation and storage, and improve process effect

Inactive Publication Date: 2018-05-04
SUZHOU KUANGSHI JUNCHI BIOLOGICAL SCI & TECH
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Problems solved by technology

[0004] The purpose of the present invention is to provide a preparation method of organic alcohol oxidase, aiming at the defect of poor thermal stability of existing organic alcohol oxidase, the developed organic alcohol oxidase comes from the thermophilic microorganism Aeropyrum pernix, and has been genetically modified , so that it can exert the highest activity in the temperature range of 50-55 degrees, and it is stable in high temperature for a long time

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  • Preparation method of organic alcohol oxidase

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Embodiment

[0023] A preparation method of organic alcohol oxidase, comprising the following steps:

[0024] Step 1. Design and synthesize the organic alcohol oxidase gene of the thermophilic microorganism Aeropyrum pernix, and insert it into the pET28 expression vector to construct a recombinant expression plasmid of DNA ligase; Continuous histidine affinity purification tags for immobilized nickel ion affinity chromatography purification;

[0025] Step 2. Recombinant expression of thermostable organic alcohol oxidase; transform the recombinant expression plasmid of thermophilic microorganism Aeropyrum pernix organic alcohol oxidase into Escherichia coli expression host BL21 (DE3), and obtain the recombinant expression strain of thermostable organic alcohol oxidase; express Strains were grown to OD 600 =0.6, add 0.5mM inducer IPTG, culture at 20°C for 12 hours, induce the expression of thermostable organic alcohol oxidase;

[0026] The amino acid sequence (nitrogen terminal → carbon te...

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Abstract

The invention discloses a preparation method of organic alcohol oxidase. The preparation method comprises the following steps: 1) designing and synthesizing a thermophile Aeropyrum pernix organic alcohol oxidase gene, and interpolating the gene into a pET28 expression vector, so that a recombinant expression plasmid of DNA ligase is constructed; 2) promoting recombinant expression of thermal-stable organic alcohol oxidase; 3) conducting affinity purification of the thermophile Aeropyrum pernix organic alcohol oxidase; and 4) detecting temperature dependency of activity of the thermophile Aeropyrum pernix organic alcohol oxidase. According to the preparation method provided by the invention, the optimum enzymatic activity pH of the prepared alcohol oxidase is 7.0, temperature is at 50 DEG Cand thermal stability is that a half-life period is 10h at 70 DEG C; and the preparation method is applicable to enzymatic preparation of organic aldehyde.

Description

technical field [0001] The invention relates to a preparation method of organic alcohol oxidase. Background technique [0002] At present, the preparation of organic aldehyde pharmaceutical intermediates mostly adopts chemical catalysis, which has many by-products, difficult separation and purification, and heavy pollution. The form of increasingly emphasizing environmental protection has become increasingly unsuitable for the development of my country's pharmaceutical intermediate industry. In such a form, the preparation of organic aldehydes by bio-enzyme catalysis has increasingly become the main process. The biological enzymes currently used to oxidize organic alcohols into organic aldehydes are not very good, especially the ability to tolerate organic solvents and thermal stability are relatively poor. [0003] The currently used organic alcohol oxidase is derived from microorganisms at room temperature, which can only have the highest activity at 30-40 degrees, and i...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/70
CPCC12N9/0006C12N15/70
Inventor 刘喜朋
Owner SUZHOU KUANGSHI JUNCHI BIOLOGICAL SCI & TECH
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