Thermostable citrate synthase gene, engineered bacterium containing gene and expression method thereof

A technology of citric acid synthase and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problems of unfavorable industrial production, high fermentation temperature, and high manufacturing cost, and achieve the effects of saving production cost, heat-resistant enzyme activity, and saving production conditions

Inactive Publication Date: 2019-01-25
ANHUI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The optimal fermentation temperature of citric acid synthase produced by citric acid is relatively high. During the fermentation process, a large amount of cooling water is required to cool the fermenter to maintain the normal fermentation temperature. It is also difficult to achieve, resulting in high manufacturing costs, low output, and poor safety, which is not conducive to large-scale industrial production

Method used

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  • Thermostable citrate synthase gene, engineered bacterium containing gene and expression method thereof
  • Thermostable citrate synthase gene, engineered bacterium containing gene and expression method thereof
  • Thermostable citrate synthase gene, engineered bacterium containing gene and expression method thereof

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Embodiment 1

[0032] A heat-resistant citrate synthase gene, the heat-resistant citrate synthase gene has the base sequence of SEQ ID NO.1.

[0033] A genetic engineering bacterium of heat-resistant citrate synthase, the construction method of the engineering bacterium comprises primer design, construction of a recombinant expression plasmid and host bacterium transformation, the specific steps are as follows:

[0034] 1) Primer design: According to the known Anabaena sp.PCC7120 citrate synthase gene (NC_003272.1), a pair of forward primers gltAF containing an Nde I site and an Xho I site were designed The primer of the reverse primer gltAR, wherein, the sequence of the forward primer gltAF: 5'-AAATGTGCATATGATGGTGTGCGAATACAAGCCTG-3', the reverse primer gltAR: 5'-AGCCGCTCGAGTTATTTCCCCAGCTTTTAACGTTGGTCAA-3';

[0035] 2) Constructing a recombinant expression plasmid: using the whole genome DNA of Anabaena PCC 7120 as a template, using PCR amplification technology to obtain the target gene frag...

Embodiment 2

[0040]1. Determination of thermostable citrate synthase (CS): citrate synthase can catalyze the condensation of acetyl coenzyme A and oxaloacetate to generate citric acid and coenzyme A, and its activity determination method refers to the DTNB method of Srere et al. DTNB (5,5'-dithio-2-nitrobenzoic acid) is Ellman's reagent. In the presence of sulfur compounds, colorless DTNB will be transformed into yellow 5-mercapto-2-nitrobenzoic acid . Since 5-mercapto-2-nitrobenzoic acid has a maximum absorption peak at 412nm, the enzyme activity can be determined by monitoring the change of A412nm. The determination method is as follows:

[0041] Reaction system: Tris-HCL (pH 8.0), oxaloacetate, acetyl-CoA, DTNB.

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Abstract

The invention discloses a thermostable citrate synthase gene, engineering bacteria containing the gene and expression method thereof, in accordance with that present invention, the gene of the heat-resistant citrate synthase of the invention takes the genome of Anabaena sp. PCC7120 as a template and is obtained by using PCR amplification technique, the recombinant plasmid containing citrate synthase gene was transformed into Escherichia coli, after kanamycin resistance screening and sequencing, the citrate synthase produced by the genetic engineering bacteria has the characteristics of heat resistance and high enzyme activity, which provides a basis for its better application in the industrial production of citric acid. The characteristics of heat resistance and high enzyme activity enableit to better save production costs.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a heat-resistant citrate synthase gene, an engineering bacterium containing the gene and an expression method thereof. Background technique [0002] Citrate synthase (CS, EC 4.1.3.7) exists in almost all organisms, and it is a key rate-limiting enzyme of many important metabolic pathways in cells and a marker enzyme of metabolic changes. In the TCA cycle, citrate synthase can catalyze the condensation reaction between oxaloacetate and acetyl-CoA to generate citrate and CoA. Citric acid is a very important organic acid, known as the largest organic acid, is widely used in food, environmental protection, chemical, textile and pharmaceutical, animal husbandry and other industries, and the demand is increasing year by year. my country is the world's largest producer of citric acid, accounting for more than 70% of the world's production capacity and about 65% ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N1/21C12N15/70C12N15/66C12N9/88
CPCC12N9/88C12N15/66C12N15/70C12Y401/03
Inventor 葛亚东蒋璐璐侯少林苏凤智
Owner ANHUI NORMAL UNIV
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