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Genetic engineering bacteria producing glycerol dehydrogenase (GDH) and GDH preparation method

A technology of genetically engineered bacteria and glycerol dehydrogenase, which is applied in the field of GDH-producing genetically engineered bacteria and its GDH preparation, can solve the problems that the cost and production capacity cannot reach industrial production, and achieve the effect of low cost

Inactive Publication Date: 2009-04-29
HUAQIAO UNIVERSITY
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Problems solved by technology

[0005] GDH, GDHt, and PDOR are encoded by different genes on the dha operon. Some people have used genetic engineering techniques to transform the strains. Ichinose et al. introduced glycerol dehydrogenase from Escherichia coli strain JM109 genomic DNA in Escherichia coli expression, but in terms of cost and production capacity, it cannot meet the requirements of industrial production

Method used

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  • Genetic engineering bacteria producing glycerol dehydrogenase (GDH) and GDH preparation method

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Embodiment 1

[0012] Construction of recombinant engineering bacteria producing GDH: Total DNA was extracted from Klebsiella pneumoniae DSM 2026 strain by boiling method, with 5′-CGTCGGATCCTACATGCGCACTTATTTGAG-3′ as the front primer and 5′-AATGCTCGAGCGAATTAACGCGCCAGCCAC-3′ as the back primer, and PCR amplification was used to obtain For the target gene gldA, refer to the third edition of the "Molecular Cloning Experiment Guide" written by American Sambrook et al. Insert the target gene into pMD18-T to obtain the recombinant plasmid pMD-gldA. Digested with Xho I, designed a ligation system according to the instructions of T4 DNA Ligation (Biolab), and ligated to obtain the recombinant plasmid pET-32gldA. Specific process such as figure 1 shown.

Embodiment 2

[0014] Cultivation of recombinant engineered bacteria: transfer the recombinant plasmid pET-32gldA into E. coli strain BL21 by thermal transformation method, select the transformants and insert them into 5 mL of LB medium containing 75 μg / mL ampicillin, and culture them in a shaker at 37°C until For several phases, transfer 1-5% of the inoculum to 50 mL of LB medium containing 75 μg / mL ampicillin, shake and culture at 200 rpm, 30-37 °C for 0.5-3 h, and add the inducer lactose until the end. The concentration is 0.5-20mg / mL to induce expression.

Embodiment 3

[0016] Preparation and activity detection of recombinant glycerol dehydrogenase: suspend genetically engineered bacteria in phosphate buffer (containing dithiothreitol), and ultrasonically disrupt cells in an ice bath; remove insoluble cell debris to obtain a cell-free extract, according to Nickel column affinity chromatography is carried out by a conventional method to obtain the recombinant glycerol dehydrogenase.

[0017] GDH dehydrogenates glycerol to dihydroxyacetone, coenzyme I NAD + The hydrogen obtained as an acceptor is reduced to NADH, which has a maximum absorption under ultraviolet light at 340nm. Therefore, according to the increase of absorbance, the activity of GDH was determined by the initial velocity method. Cultured in a 250mL shake flask, the specific activity of GDH obtained after separation and purification is 188U / mg, which is about 2.7 times that of GDH expressed by Keiko in E.coliHB101 / pSE420D, which is 2.7 times higher than that obtained by Chen Hong...

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Abstract

The invention belongs to the field of biotechnology, which comprises the following steps: adopting a PCR technology to clone a gldA gene from klebsiella pneumoniae DSM 1115, constructing an Escherichia coli expression vector pET-32gldA containing the gldA gene and transforming the Escherichia coli expression vector to Escherichia coli BL21 (DE3) so as to obtain a recombinant genetic engineering strain E.coli-pET-gldA which can express glycerol dehydrogenase (GDH); and culturing recombinant bacteria to OD 0.4-1.2, and obtaining recombinant glycerol dehydrogenase through lactose-induced expression. The invention can be applied to producing dihydroxyacetone which is widely used in medicine, pesticides, cosmetics and food additives through an enzymatic process.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and in particular relates to a genetic engineering bacterium producing glycerol dehydrogenase and a preparation method thereof, that is, a genetic engineering bacterium producing GDH and a preparation method thereof. Background technique [0002] Dihydroxyacetone, English name dihydroxyacetone, abbreviated as DHA, is the simplest ketose, soluble in water and organic solvents such as ethanol, acetone, ether, etc., and stable at pH 6.0. There are very few reports about DHA in China, and DHA has been widely used in foreign countries: (1) DHA molecule contains three functional groups, has active chemical properties, and widely participates in various chemical reactions such as polymerization and condensation. It is an important chemical compound. The synthetic intermediate is also an important multifunctional reagent. For example, in industrial applications, it can ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/70C12N9/04C12R1/19
Inventor 方柏山张婷婷
Owner HUAQIAO UNIVERSITY
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