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Bacterial strain for producing reductive dehalogenase and application thereof

A technology of dehalogenase and strain, applied in the directions of enzymes, bacteria, enzymes, etc., can solve the problems of high requirements for equipment and operating conditions, difficult safe operation, low total yield, etc., achieving simple medium components and avoiding reaction time. The effect of long and simple reaction process

Active Publication Date: 2021-03-30
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical synthesis faces the problems of long reaction time, high energy consumption, toxic by-products and environmental pollution
For example, the hydrogen reduction method of 2,3,6-trichloropyridine has high requirements on equipment and operating conditions, and it is difficult to operate safely, so it is not suitable for general enterprises.
Synthetic method of 2-chloropyridine to obtain a mixture of 2,3-dichloropyridine and 2,5-dichloropyridine, difficult post-processing and low overall yield
There is no report about the catalytic synthesis of 2,3-dichloropyridine using reductive dehalogenase

Method used

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  • Bacterial strain for producing reductive dehalogenase and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Classification of Pseudomonas strains

[0017] 1) Strain characteristics

[0018] The colony on the preservation medium was milky white opaque, nearly round, with a smooth and moist surface and regular edges; observed under a microscope, the bacteria were rod-shaped and Gram-negative; the physiological and biochemical indicators are shown in Table 1.

[0019] Table 1 Physiological and biochemical characteristics of strain YtRDases-55

[0020] project YtRDases-55 project YtRDases-55 Oxidase + VP test + H2S production - MR test - Nitrate reduction + indole production - Urease - Starch Utilization - gelatin liquefaction + Catalase +

[0021] 2) Strain identification

[0022] The length of the 16S rRNA gene sequence obtained by DNA extraction, PCR amplification and sequencing was 1439 bp, and the BLAST comparison was performed on GenBank and the phylogenetic tree was drawn ( figure 1 ), the base ...

Embodiment 2

[0031] 1. Activation of the strains: pick the strains and inoculate them on the slant medium: glucose 1g / L, yeast extract powder 5 g / L, peptone 10 g / L, NaCl 5 g / L, agar 20 g / L, pH7. 0, cultured in a constant temperature incubator at 30°C for 16 hours;

[0032] 2. Liquid fermentation: Pick a circle of activated strains on the slant and inoculate them in a 500mL Erlenmeyer flask with 50mL of fermentation medium at a speed of 200r / min and a temperature of 30°C to obtain a fermented liquid of reductive dehalogenase. Culture for 72h at 8000r / m min centrifuged for 20min to collect bacterial cells, the composition and final concentration of the fermentation medium were: glucose 5 g / L, 2,3,6-dichloropyridine 0.5 g / L, (NH 4 ) 2 SO 4 5g / L, KH 2 PO 4 1g / L, MgSO 4 0.2g / L, vitamin B 1 1mg / L, biotin 50μg / L, nicotinamide 1mg / L, calcium pantothenate 1mg / L, FeSO 4 1.6 mg / L, CuSO 4 94μg / L, ZnSO 4 0.23mg / L, sodium molybdate 0.17mg / L, boric acid 50μg / L, MnSO 4 0.38 mg / L, pH 7.0-7...

Embodiment 3

[0035] 1. Strain activation: pick the strains and inoculate them to the slant medium: glucose 1g / L, yeast extract powder 5 g / L, peptone 10 g / L, NaCl 5 g / L, agar 20 g / L, pH 7.0 , cultivated in a constant temperature incubator at 30°C for 16 hours;

[0036] 2. Liquid fermentation: Pick a circle of activated strains on the slant and inoculate them in a 500mL Erlenmeyer flask with 50mL of fermentation medium at a speed of 200r / min and a temperature of 30°C to obtain a fermented liquid of reductive dehalogenase. Culture for 72h at 8000r / m min centrifuged for 20min to collect bacterial cells, the composition and final concentration of the fermentation medium were: glucose 5 g / L, 2,3,6-dichloropyridine 0.5 g / L, (NH 4 ) 2 SO 4 5g / L, KH 2 PO 4 1g / L, MgSO 4 0.2g / L, vitamin B 1 1mg / L, biotin 50μg / L, nicotinamide 1mg / L, calcium pantothenate 1mg / L, FeSO 4 1.6 mg / L, CuSO 4 94μg / L, ZnSO 4 0.23mg / L, sodium molybdate 0.17mg / L, boric acid 50μg / L, MnSO 4 0.38 mg / L, pH 7.0-7.2. ...

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Abstract

The invention relates to a strain for producing reductive dehalogenase and application of the strain, and belongs to the technical field of biology. The strain is pseudomonas YtRDases-55 and is classified and named as pseudomonas sp., the preservation unit is China General Microbiological Culture Collection Center, the preservation address is Beijing, the preservation number is CGMCC No. 21296, and the preservation date is December 4, 2020. The pseudomonas YtRDases-55 provided by the invention is good in hereditary stability, simple in culture medium component and low in raw material cost; theproduced reductive dehalogenase is high in activity, strong in substrate specificity and high in substrate conversion rate, and the strain can be directly used for catalyzing 2,3,6-trichloropyridineto dechloridize and synthesize 2,3-dichloropyridine in a whole-cell manner; in conclusion, the pseudomonas YtRDases-55 has industrial application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a bacterial strain producing reductive dehalogenase and a method for catalyzing and preparing 2,3-dichloropyridine using the reductive dehalogenase produced by the strain. Background technique [0002] 2,3-Dichloropyridine (2,3-dichloropyridine), white powdery solid, slightly soluble in water, is the key intermediate of the new insecticides chlorantraniliprole and cyantraniliprole, widely used in medicine and pesticides Research areas. With the growing market of chlorantraniliprole and cyantraniliprole, the demand for 2,3-dichloropyridine, the key intermediate of the above two pesticide products, will increase significantly, and the market prospect is broad. At present, the way to obtain 2,3-dichloropyridine is mainly based on chemical synthesis. Chemical synthesis faces the problems of long reaction time, high energy consumption, toxic by-products and environmental pol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/14C12P17/12C12R1/38
CPCC12N1/20C12N9/14C12P17/12C12Y308/01
Inventor 冯志彬张娟燕蕊陈泉炎程仕伟
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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