Quantitative screening method for halohydrin dehalogenase library and related microfluid system

A technology of halohydrin dehalogenase and microfluidic system is applied in the field of enzyme engineering to achieve the effects of simple detection process, low price and easy acquisition

Inactive Publication Date: 2019-07-26
UNIV OF ELECTRONICS SCI & TECH OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the problems existing in the existing library screening methods, the present invention provides a method for rapid and quantitative screening of halohydrin dehalogenases and related microfluidic systems based on paper-based microfluidic platforms through image analysis of color changes caused by pH changes

Method used

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  • Quantitative screening method for halohydrin dehalogenase library and related microfluid system
  • Quantitative screening method for halohydrin dehalogenase library and related microfluid system
  • Quantitative screening method for halohydrin dehalogenase library and related microfluid system

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Experimental program
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Effect test

Embodiment 1

[0051] System settings and reagent selection of the microfluidic platform;

[0052] Step A: Selection of carrier;

[0053] The specific steps include: screening 7 different types of cellulose and glass fiber membrane paper on the market as the preferred carrier material. In this embodiment, the neutral carrier is selected. The material is simple and low in cost, making it an ideal carrier material.

[0054] Step B: indicator / buffer system

[0055] The choice of indicator depends on the optimal pH of enzyme activity. The optimal pH of halohydrin dehalogenase is 8-9, and the p Ka value of indicator and buffer should be similar. Therefore, phenol red (pKa = 8.0) and HEPES (pKa = 8.2) were selected as indicators and buffers in the catalytic reaction, respectively; an equal amount of HEPES buffer dissolved in 8 μl of phenol red solution was added dropwise to 7 discs (from From left to right are marked as A to G). Use a 6mm punching machine to punch the above seven filter paper carriers, a...

Embodiment 2

[0063] Screening mutation libraries

[0064] Step A: Protein expression and collection

[0065] The wild-type halohydrin dehalogenase and the heterologous expression mutant in recombinant E. coli were selected, cultured at 120 rpm at 37°C for 18 hours and suspended in 10 mM HEPES, and the cells were lysed by ultrasonic treatment. After centrifugation, the supernatant was collected as a crude enzyme extract. The Bradford method was used to determine the protein concentration, and the halide release method was used to measure the activity; for whole cell screening, the cells were suspended in HEPES buffer, washed twice, and used for library screening; The components of the medium used include 10g / L peptone, 5g / L yeast extract, 5g, 10g / L sodium chloride, ampicillin as an antibiotic, and arabinose as an inducer;

[0066] Step B: Library screening of cell-free and whole-cell enzymes

[0067] In order to prove the utility of the present invention for library screening, 5mM 1,3-DCP was used...

Embodiment 3

[0069] Activity analysis of halohydrin dehalogenase mutants

[0070] Four substrates of 1,3-DBP, 2,3-DBP, 1,3-DCP and 2,3-DCP were used to determine the enzyme activity of the HheC mutant; first use a 6mm puncher to punch out 5 paper discs and place them Into a petri dish, two of the paper discs are used as negative controls (without transferring the halohydrin dehalogenase genome) and blank (without enzyme), and the other three paper discs are added with the same enzyme, the same substrate and the color reagent . Fix the camera on a tripod to capture images, and take images every 30 seconds for 10 minutes. With the increase of the reaction product concentration, the color of the filter paper changes continuously, and the steepest part of the fitting curve is used to determine the initial rate; the reaction difference of different substrates is studied by changing the type of substrate in the paper tray, and the result is Figure 4 As shown, the results indicate that the HheC mu...

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Abstract

The invention discloses a quantitative screening method for a halohydrin dehalogenase library and a related microfluidic system, and belongs to the technical field of enzyme engineering. Halohydrin dehalogenase is utilized to catalyze a substrate to release protons to cause pH reduction, the pH reduction can be reflected by color change of an indicator, and the microfluidic system integrating image acquisition and image analysis equipment is built on the basis of a colorimetric principle. The quantitative screening method for the library comprises the following steps: based on a paper microfluidic platform, adsorbing halohydrin dehalogenase on a microfluidic carrier fixed with the indicator; adding the substrate dropwise, then incubating, and detecting the change of the pH value through the indicator; after the color changes, acquiring images, acquiring a linear relationship between proton concentration and the color intensity of the indicator through image analysis as a standard curve; detecting proton concentration in a microfluidic reaction system adsorbing a halohydrin dehalogenase mutant based on the standard curve, and determining enzyme activity by the proton concentration in the microfluidic reaction system. The quantitative screening method for the halohydrin dehalogenase library and the related microfluidic system has the characteristics of simplicity, rapidness and sensitivity, and quantitative screening of the halohydrin dehalogenase library is realized.

Description

Technical field [0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to a method for quantitative screening of a halohydrin dehalogenase library and a related microfluidic system. Background technique [0002] Halohydrin dehalogenases (HHDHs) are important enzymes in the field of biotechnology. They can catalyze the dehalogenation of halohydrins to generate epoxides, protons and halide ions. The halohydrin dehalogenase HheC from A.radiobacter AD1 and the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can be used together in the biodegradation process of 1,2,3-TCP. It is an effective means to degrade organic halides. . In addition, HHDHs have good stereo and regioselectivity, can be used to produce valuable chiral β-substituted alcohols, and have attracted much attention in the field of compound synthesis. So far, halohydrin dehalogenases have been successfully used in the synthesis of chiral epoxides, halohyd...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/34C12M1/00C12Q1/34C40B30/08
CPCC12Q1/34C12Q2334/70C40B30/08
Inventor 汤丽霞伊嘉格尔陈勇邓娇方瑞琴
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA
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