Method for producing alanine from recombinant Escherichia coli by the aid of glycerin

A technology of Escherichia coli and alanine, applied in the field of microbial genetic engineering, can solve the problems of long production cycle, increased food cost, large equipment investment, etc., and achieve the effect of reduced production cost and stable output

Inactive Publication Date: 2015-04-29
CHANGZHOU QIUHONG BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the concentration of the product is low, the production cycle is long, the investment in equipment is large, there are many by-products, and the separation is complicated
And fermentation uses sugary resources, but today, food resources are increasingly tight, the process of fermenting alanine with sugary resources as raw materials is not only complicated in production process, but also the cost of food is increasing day by day

Method used

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  • Method for producing alanine from recombinant Escherichia coli by the aid of glycerin

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Embodiment 1

[0057] 1) Cloning of gene fragments

[0058] Using primer sequence 1 and primer sequence 2, the gene fragment alaD was amplified, and the PCR amplification conditions were: 95°C for 30S, 62°C for 20S, 72°C for 30S, 30 cycles, and 72°C for 10 min.

[0059] 2) Construction of plasmid

[0060] The amplified gene fragment was digested with different restriction enzymes KpnI and XbaI to obtain cohesive ends, and the pUC19 vector was also digested with restriction enzymes KpnI and XbaI. The digestion conditions were all at 37°C and incubated for 2 hours. Recover all the obtained gene fragments and linearized vectors, mix the purified gene fragments and linearized vectors at equal concentrations, add ligation buffer and DNA ligase, and connect overnight at 37°C; obtain plasmid pALA ligation solution ;

[0061] 3) Screening of positive clones

[0062] The obtained plasmid pALA connection solution was transformed into E. coli cells by heat shock method. The specific method was as f...

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Abstract

The invention relates to a method for producing alanine from recombinant Escherichia coli by the aid of glycerin. The method comprises steps as follows: 1) cloning a gene alaD involved in biosynthesis of the alanine; 2) connecting the gene with an Escherichia coli high-copy vector pUC19 to construct a recombinant vector of a coding gene containing protein required by biosynthesis of the alanine; 3) transferring a recombinant plasmid into the Escherichia coli BW25141, and obtaining a strain, which can realize the high yield of the alanine, of the Escherichia coli through selection marking by ampicillin; 4) producing the alanine through fermentation, and recovering the alanine from a fermentation liquid. The method has the advantages of environment-friendliness, high efficiency, stability, low cost and the like.

Description

Technical field: [0001] The invention belongs to the field of microbial genetic engineering. Specifically, a gene involved in the biosynthesis of alanine is cloned and connected to the high-copy vector pUC19 of E. coli, and the recombinant plasmid is transferred into E. coli, and ampicillin is used as a screening marker to enhance The expression level of the gene in Escherichia coli enables alanine to be synthesized in large quantities in Escherichia coli. Background technique: [0002] Alanine, also known as 2-aminopropionic acid, is an aliphatic nonpolar amino acid. Molecular formula is C 3 h 7 o 2 N, the structural formula is as follows, and the relative molecular mass is 89.063. The common one is L-alanine, which is one of the protein-encoded amino acids, a non-essential amino acid and a sugar-producing amino acid for mammals. Only L-alanine can be absorbed and utilized by the human body. D-alanine is present in the peptidoglycan of many bacterial cell walls or as a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/06
CPCC12P13/06C12N1/205C12R2001/19
Inventor 刘立栋
Owner CHANGZHOU QIUHONG BIOTECH
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