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Method for quickly screening oxygenase microbes or oxygenase genes

A technology of oxygenase and microorganisms, applied in the field of microorganisms, can solve the problems of screening natural source microorganisms from environmental samples that have not been reported yet, and achieve the effect of improving screening sensitivity and high molar absorptivity coefficient

Inactive Publication Date: 2011-01-12
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the directed evolution study of the recombinase cytochrome P4502A6, a method for screening mutants using 4-chloroindole was reported, but so far, there has been no report for screening natural microorganisms from environmental samples

Method used

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  • Method for quickly screening oxygenase microbes or oxygenase genes
  • Method for quickly screening oxygenase microbes or oxygenase genes
  • Method for quickly screening oxygenase microbes or oxygenase genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 takes 4-chloroindole as substrate screening producing cyclooxygenase strain

[0026] Activated sludge samples were taken from a sewage treatment plant, enriched in a medium containing M12 inorganic salts and styrene (concentration: 2mM), and cultured at 30°C and 225rpm for 10 days. Dilute the enriched culture solution onto a screening plate (M12+styrene 2mM+4-chloroindole 0.1mM+0.01% yeast powder+1.5% agar powder) and culture at 30°C for 3 days, multiple flakes can be seen on each plate blue plaque ( figure 1 ), pick out and continue to purify ( figure 2 ), and 11 pure strains were obtained.

Embodiment 2

[0027] Example 2 Using 4-methylindole as a substrate to screen cyclooxygenase-producing strains

[0028] The sample and enrichment method are the same as in Example 1, and the enriched culture solution is diluted onto a screening plate (M9+styrene 2mM+4-methylindole 0.2mM+0.05% yeast powder+1.5% agar powder) and cultured at 30°C for 3 One day, blue plaques appeared, and the purification was continued to obtain 15 pure strains.

[0029] Example 3 Using 4-indolecarboxylate as a substrate to screen cyclooxygenase-producing strains

[0030] The sample and enrichment method are the same as in Example 1. Dilute the enriched culture solution onto a screening plate (M9+styrene 3mM+4-indolecarboxylate methyl ester 0.3mM+0.06% yeast powder+1.5% agar powder) and culture at 30°C After 4 days, blue plaques appeared, and the purification was continued to obtain 12 pure bacterial strains.

Embodiment 4

[0031] Embodiment 4 uses 4-bromoindole as substrate screening to produce cyclooxygenase strain

[0032] Take from another sewage treatment plant sample, the enrichment method is the same as in Example 1, and the enrichment culture solution is diluted onto the screening plate (M9+styrene 3mM+4-bromoindole 0.2mM+0.05% yeast powder+1.5% agar powder ) was cultured at 30°C for 4 days, dark blue plaques appeared, and the purification was continued to obtain 10 pure strains.

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Abstract

The invention belongs to the technical field of microbes, and particularly relates to a method for quickly screening oxygenase microbes or oxygenase genes. The method for quickly screening oxygenase microbes comprises the following steps: using the only carbon source enriched environmental sample, coating the enriched environmental sample on the culture medium, culturing, separating and purifying to obtain the microbe strain of the target oxygenase. The method for quickly screening oxygenase genes comprises the following steps: constructing a metagenomic library of environmental samples, and screening the oxygenase gene from the metagenomic library. The invention has the advantage of high screening sensitivity, and can be used for conveniently and quickly screening oxygenase resources in the environmental sample in a high flux mode.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for rapidly screening oxygenase microorganisms or oxygenase genes. Background technique [0002] Biocatalysis has become the development trend of today's chemical production and related technical fields due to its high chemical, regio, and stereoselectivity, mild reaction conditions, easy post-treatment, and low energy consumption, and has been successfully applied in many fields. Biological enzymes are the core of biocatalytic technology, and the acquisition of new and efficient catalytic enzymes has become a technical bottleneck in this field. At present, the development and utilization of oxygenase is still very limited, one of the important reasons is that there are many limitations in traditional oxygenase screening methods, making it difficult to screen efficient and highly selective oxygenase strains and genes. [0003] The oxygenases mentioned...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68
Inventor 吴中柳刘艳张超张志刚
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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