A rapid screening method for halohydrin dehalogenase

A technology of halohydrin dehalogenase and screening method, which is applied in the field of enzyme screening, can solve the problems of long period, heavy workload, high cost, etc., and achieve the effect of low cost, fast speed, and less culture medium and reagents

Active Publication Date: 2017-05-24
NANJING NUOYUN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the problems of high cost, heavy workload and long cycle of existing methods for screening halohydrin dehalogenases.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1）

[0036] 1. Preliminary screening of halohydrin dehalogenase

[0037] Pick a single colony of Escherichia coli containing the halohydrin dehalogenase gene (well No. 1 is the original control), and inoculate it in the wells of a 48-well deep-well plate containing 0.6ml of ampicillin-resistant liquid medium. The volume is 4.6ml. Cover the sandwich lid of the deep-well plate, incubate at 37°C, 200rpm shaker for 6h, add IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 1mM, and adjust the temperature to 30°C , 200rpm shaker to continue culturing for 20h.

[0038] The medium used is: peptone 10g / L, yeast powder 5g / L, glycerin 10g / L, potassium dihydrogen phosphate 1.0g / L, magnesium sulfate 0.2g / L, pH 7.0, 0.1MPa sterilization for 30min, to be cooled Add ampicillin to room temperature to a final concentration of 100 μg / mL.

[0039] After the end, put the deep-well plate into the ‐80°C ultra-low temperature refrigerator. After the temperature is stable, take the ...

Embodiment 2）

[0055] 1. Preliminary screening of halohydrin dehalogenase

[0056] A single colony of Escherichia coli containing the halohydrin dehalogenase gene was picked and inoculated in the wells of a 48-well deep-well plate containing 0.6 ml of ampicillin-resistant liquid medium, and the volume of each well was 4.6 ml. Cover the sandwich lid of the deep-well plate, incubate at 37°C, 200rpm shaker for 6h, add IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 1mM, and adjust the temperature to 30°C , 200rpm shaker to continue culturing for 20h.

[0057] The medium used is: peptone 10g / L, yeast powder 5g / L, glycerin 10g / L, potassium dihydrogen phosphate 1.0g / L, magnesium sulfate 0.2g / L, pH 7.0, 0.1MPa sterilization for 30min, to be cooled Add ampicillin to room temperature to a final concentration of 100 μg / mL.

[0058] After the end, put the deep-well plate into the ‐80°C ultra-low temperature refrigerator. After the temperature is stable, take the deep-well plate...

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Abstract

The invention relates to an enzyme screening method, particularly to a screening method for a halohydrin dehalogenase. Halohydrin dehalogenase with high enzyme activity is screened by pH color development and a primary screening-secondary screening two-step method. By employing a deep-hole plate for microminiaturized culture, the invention realizes high throughput culture and high throughput preparation of a crude enzyme liquid. PH change is utilized as a breakthrough to screen halohydrin dehalogenase with high enzyme activity, the efficiency is high, the cost is low, and the speed is fast. Fewer media and reagents are consumed. The method provided by the invention makes mechanized automatic operation come true, and with the help of an automated high-throughput screening instrument, the amount of labor can be greatly reduced further, the screening flux can be improved.

Description

technical field [0001] The invention relates to an enzyme screening method, in particular to a halohydrin dehalogenase screening method. Background technique [0002] Halohydrin dehalogenase (HHDH) is a catalytically active hydrolase that can reversibly act on ortho-hydroxyhalides (ortho-halohydrins) to form epoxides and ring-opening of epoxides. [0003] Epoxides are recognized as one of the most important and widely used synthetic intermediates in organic synthesis. The chiral synthesis and ring opening of epoxides are very important for the synthesis of chiral drugs. For example, in the enzymatic synthesis of the side chain intermediate A5 of atorvastatin calcium ((R)‐4‐cyano‐3‐hydroxybutyrate ethyl ester), halohydrin dehalogenase is used as a biocatalyst. [0004] The natural halohydrin dehalogenase has high production cost, low enzyme activity, and poor stability in the industrialization process, which restricts the application of this type of enzyme in large-scale pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/527
Inventor 陈令伟
Owner NANJING NUOYUN BIOLOGICAL TECH CO LTD
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