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A high-throughput screening method for an L-aspartate beta-decarboxylase producing strain

A technology of aspartic acid and decarboxylase, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve the problems of decreased cell enzyme activity and increased pH value.

Active Publication Date: 2016-06-08
SHANDONG INT BIOTECH PARK DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, in the conversion process of L-alanine enzymatic method, the decarboxylation reaction makes the pH value of the reaction solution continuously rise, which deviates from the optimum pH value range of the enzyme reaction, resulting in a decrease in the enzyme activity of the cells, which has great impact on the process control and bioreactor. make higher demands

Method used

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  • A high-throughput screening method for an L-aspartate beta-decarboxylase producing strain
  • A high-throughput screening method for an L-aspartate beta-decarboxylase producing strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Screening of L-aspartic acid β-decarboxylase producing bacteria

[0023] 1. Enrichment and separation of microorganisms in samples

[0024] Enrichment medium: beef extract 0.5%, peptone 1%, yeast powder 0.2%, KH 2 PO 4 0.05%, K 2 HPO 4 ·H 2 O0.14%, NaCl1.5%, pH7.0-7.2, sterilized at 121°C for 20min, cooled for later use;

[0025] Separation medium plate: 0.5% beef extract, 1% peptone, 1.5% NaCl, 2% agar, pH8.5-9.0, sterilized at 121°C for 20min, then pour the plate to make a separation plate, 20mL / dish, cool and solidify use;

[0026] 1. Sampling

[0027] It has been reported in the literature that marine microorganisms have an ecological community structure diversity and species diversity unmatched by terrestrial microorganisms. Therefore, the Yellow Sea area of ​​Yantai was selected as the sampling site, and a total of 10 samples of sponges, corals, and seabed mud were collected.

[0028] 2. Enrichment

[0029] After the sample was taken back to the...

Embodiment 2 Embodiment 1

[0059] Example 2 Identification of the bacterial species of the L-aspartic acid β-decarboxylase producing bacteria obtained in Example 1

[0060] 1. Identification of strain morphology and physiological and biochemical characteristics

[0061] Refer to the method of "Common Bacteria System Identification Manual" to identify strains with strain morphology and physiological and biochemical characteristics

[0062] 1. Morphological characteristics of the thalline The L-aspartic acid β-decarboxylase producing bacteria obtained in Example 1 is a Gram-negative bacterium, and the cells are short rod-shaped, with extreme flagella and no spores; they can form a flat plate on the broth agar plate The round colonies are yellowish in color, with a rough surface, and the old culture has a foul smell.

[0063] 2. The identification results of physiological and biochemical characteristics are shown in Table 1.

[0064] Table 1 Physiological and biochemical characteristics of L-aspartate β-...

Embodiment 3

[0068] Example 3 Comparative example: performance investigation of biotransformation production of L-alanine

[0069] This comparative example investigated the performance difference between the test strain and the control strain for transforming and producing L-alanine. Wherein the test strain is the high-yield bacterial strain control bacterial strain Pseudomonas putida (Pseudomonasputida) screened in embodiment 1, and the control bacterial strain Pseudomonas dacunhae is purchased from China Industrial Microorganism Culture Collection Management Center, numbering 1511C0005000003137, and this bacterial strain is the production of L-alanine strain.

[0070] 1. Shake flask fermentation culture

[0071] Shake flask fermentation medium formula: glucose 3%, peptone 1%, yeast powder 0.2%, NaCl 1.5%, KH 2 PO 4 0.05%, K 2 HPO 4 ·H 2 O0.14%, MgSO 4 ·7H 2 O0.03%, CaCl 2 0.05%, pH6.5-7.0. The shake flask fermentation medium was sterilized at 121°C for 20 minutes, after cooling...

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Abstract

A high-throughput screening method for an L-aspartate beta-decarboxylase producing strain is disclosed. The method includes 1) subjecting microorganisms in a sample to enrichment culture and primary separation by adopting an enrichment culture medium, 2) inoculating single colonies obtained through separation from a plane plate to a 96-micropore plate primary screening semisolid medium and performing primary screening to obtain L-aspartate beta-decarboxylase producing strains, 3) inoculating the strains obtained through primary screening to a 24-prore plate, performing liquid culturing, and performing secondary screening to obtain the L-aspartate beta-decarboxylase producing strain with high yield by combining paper chromatography, and 4) subjecting the high-yield strain obtained through secondary screening to fermentation in a shake flask, and determining enzyme activity by combining an oxidation coloration process. The method is suitable for high-throughput screening of the L-aspartate beta-decarboxylase producing strain. The strain obtained through screening is alkali-resistant, thus avoiding tedious pH value controlling steps in a cell production processes. References are provided for high-throughput screening of producing strains bio-enzymes of the same kind.

Description

technical field [0001] The invention belongs to the field of microbial pharmacy, and relates to a high-throughput screening method for bacteria producing L-aspartic acid β-decarboxylase. Background technique [0002] L-aspartic acid β-decarboxylase (Asd), also known as L-aspartic acid 1-decarboxylase, can catalyze the removal of the carboxyl group of L-aspartic acid to generate L-alanine (L-Ala ). L-alanine is widely used in medicine and food industry. With the increase of demand and the development of fermentation industry, the production method of L-alanine has developed from protein hydrolysis extraction and fermentation to enzymatic production. That is, L-aspartic acid is decarboxylated to L-alanine by L-aspartic acid β-decarboxylase. The production of L-Ala by Asd transformation can be divided into free cell method and immobilized cell transformation method. Japan has fixed Pseudomonas.dacunhae cells with carrageenan in 1982 for continuous production of L-Ala by L-As...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/527C12Q1/04C12R1/40
Inventor 李东升傅风华张淑敏杨小平
Owner SHANDONG INT BIOTECH PARK DEV
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