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A method for dual fluorescence screening of β-alanine synthetase

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 fluorescence background interference value and great influence of bacterial growth status, etc., and achieve efficient and accurate detection , good application prospects, and the effect of improving detection efficiency

Active Publication Date: 2022-06-21
ZHEJIANG UNIV OF TECH
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  • Claims
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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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  • A method for dual fluorescence screening of β-alanine synthetase
  • A method for dual fluorescence screening of β-alanine synthetase
  • A method for dual fluorescence screening of β-alanine synthetase

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Determination of Bacillus subtilis-derived β-alanine synthase (PanD Bsu ) specific vitality

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

[0037] 1) The pGLO plasmid containing the gene encoding green fluorescent protein (gfp) is digested 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 an arabinose promoter was obtained by means of total gene synthesis, and connected to the universal vector pUC57 plasmid, denoted as pUC57-panD Bsu , and the nucleotide sequence is shown in SEQ ID NO.2.

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

[0040] 4) The step 1) linearized vector is ligated with the step 3) panD gene de...

Embodiment 2

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

[0058] 1. Mutation library construction

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

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Abstract

The invention discloses a method for screening β-alanine synthetase by a double fluorescence method. The fluorescent reporter gene and the β-alanine synthetase gene are jointly introduced into the host bacteria, and the basic salt medium containing arabinose is inoculated. After induction culture at 30-37°C, obtain the fermentation liquid; take the fermentation liquid and freeze-thaw the cells, and take the cell-cracked liquid as the crude enzyme liquid; add L-aspartic acid to the crude enzyme liquid, and let it stand at 37°C for 2 hours Carry out the conversion reaction and convert the substrate into β-alanine, take the conversion liquid and measure the fluorescence value under the conditions of absorption wavelength 355nm and emission wavelength 445nm; get the fermentation broth and measure the fluorescence value under the conditions of absorption wavelength 395nm and emission wavelength 509nm, High-activity β-alanine synthetase is obtained by screening; the system of the present invention has the advantages of simple operation, stable enzyme activity measurement, and high sensitivity of enzyme activity detection, and has important application value in the directional transformation of β-alanine synthase.

Description

technical field [0001] The present invention relates to the fields of genetic engineering and enzyme engineering, and more particularly, to the construction of a high-throughput screening system for β-alanine synthase. Background technique [0002] β-Alanine is mainly used in the synthesis of calcium pantothenate, carnosine, sweeteners, water purification flocculants, electroplating corrosion inhibitors, etc. β-Alanine and its derivatives are widely used in medicine, beauty, food, feed, chemical and other fields, and the market demand is increasing day by day. At present, β-Ala is mainly synthesized by chemical methods. However, the chemical synthesis method has relatively high technological requirements, produces a large number of "three industrial wastes" such as nitrile by-products and inorganic salts, and has many side reactions, which is not conducive to the separation and purification of products. The biological enzymatic production of β-alanine is more environmental...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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