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Method for screening beta-alanine synthetase by double fluorescence

A technology for alanine and synthetase, which is applied in the field of high-throughput screening system construction of β-alanine synthase, can solve the problems of large influence of bacterial growth state and large fluorescence background interference value, and achieve the goal of reducing interference Effect

Active Publication Date: 2019-06-14
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the PanD enzyme activity measured by this method is the total enzyme activity of the fermentation broth, which is greatly affected by gene expression conditions and bacterial growth status.
In addition, the culture medium used in this method is a rich medium, and the resulting fluorescence background interference value is relatively large

Method used

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  • Method for screening beta-alanine synthetase by double fluorescence
  • Method for screening beta-alanine synthetase by double fluorescence
  • Method for screening beta-alanine synthetase by double fluorescence

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: Determination of Bacillus subtilis source β-alanine synthetase (PanD by double fluorescence method Bsu ) specific activity

[0036] 1. pGLO-P ara -panD plasmid map as figure 1 As shown, its construction method is as follows:

[0037] 1) Digest the pGLO plasmid containing the gene encoding green fluorescent protein (gfp) with XbaI and KpnI to obtain a linearized plasmid vector (the nucleotide sequence is shown in SEQ ID NO.1).

[0038] 2) The panD gene derived from Bacillus subtilis containing the arabinose promoter was obtained by total gene synthesis, and connected to the universal vector pUC57 plasmid, denoted as pUC57-panD Bsu , the nucleotide sequence is shown in SEQ ID NO.2.

[0039] 3) pUC57-panD Bsu After digestion with XbaI and KpnI, a DNA fragment of the panD gene derived from Bacillus subtilis carrying an arabinose promoter (nucleotide sequence such as SEQ ID NO.3) was obtained.

[0040] 4) Ligate step 1) the linearized vector with step 3) ...

Embodiment 2

[0057] Example 2. High-throughput screening of β-alanine synthetase by dual fluorescence method

[0058] 1. Mutation library construction

[0059] It has been reported in the literature that the enzyme activity and stability of PanD derived from Bacillus subtilis are significantly better than those from other sources (Wanli Pei et al., Appl Microbiol Biotechnol, 2017, 101: 6015–6021; Tenghui Zhang et al., Process Biochemistry, 2018, 70: 117-123 ). The carrier plasmid pGLO-P carrying the Bacillus subtilis panD gene constructed in Example 1 ara -panD Bsu As a template, error-prone PCR was carried out with primers P1 and P2. The conditions of error-prone PCR are shown in Table 1 and Table 2, and the PCR product was recovered and purified by cutting the gel. Using the PCR product as a large primer, pGLO-P ara -panD Bsu The plasmid is used as a template to amplify the full-length plasmid, and the PCR product is treated with DpnI to eliminate the initial plasmid template, and t...

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Abstract

The invention discloses a method for screening beta-alanine synthetase by double fluorescence. A fluorescent reporter gene and the beta-alanine synthase gene are introduced into the host bacteria together, and a basic salt medium containing arabinose is inoculated, and the fermentation liquid is obtained after induction culture at 30 to 37 DEG C; the fermentation liquid is frozen and thawed for disruption, and disruption liquid is taken as a crude enzyme solution; the crude enzyme solution is added to L-aspartic acid, and the materials are subjected to standing at 37 DEG C for 2 h to carry outa conversion reaction to convert a substrate into beta-alanine, a fluorescence value is determined under the conditions of an absorption wavelength being 355 nm and an emission wavelength being 445 nm; the fluorescence value is measured under the conditions of the absorption wavelength being 395 nm and emission wavelength being 509 nm, and the high-activity beta-alanine synthetase is obtained byscreening. The system of the invention has the advantages of simple operation, stable enzyme activity measurement and high sensitivity of enzyme activity detection, and has important application valuein the directional transformation of beta-alanine synthase.

Description

technical field [0001] The present invention relates to the fields of genetic engineering and enzyme engineering, and more specifically, relates to the construction of a high-throughput screening system for β-alanine synthetase. Background technique [0002] β-alanine is mainly used in the synthesis of calcium pantothenate, carnosine, sweetener, water purification flocculant, electroplating corrosion inhibitor, etc. β-alanine and its derivatives are widely used in medicine, beauty, food, feed, chemical industry and other fields, and the market demand is increasing day by day. Currently, β-Ala is mainly synthesized by chemical methods. However, the process requirements of the chemical synthesis method are relatively high, and a large amount of "industrial wastes" such as nitrile by-products and inorganic salts are produced, and there are many side reactions, which are not conducive to the separation and purification of products. The production of β-alanine by biological enz...

Claims

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Application Information

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IPC IPC(8): C12Q1/527G01N21/64
Inventor 孙东昌裘娟萍毛旭丹王琳陈一扬
Owner ZHEJIANG UNIV OF TECH
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