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Construction method and application of formate dehydrogenase engineering strain

A technology of formate dehydrogenase and engineering strains, applied in the direction of microorganism-based methods, applications, genetic engineering, etc., can solve problems such as inability to meet market demand, and achieve good industrial applicability and high enzyme activity.

Active Publication Date: 2017-10-27
ECOLOGY INST SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the above-mentioned engineered bacteria can all express formate dehydrogenase, they are limited by the growth and enzyme activity of the strains and cannot meet the market demand.

Method used

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  • Construction method and application of formate dehydrogenase engineering strain
  • Construction method and application of formate dehydrogenase engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Construction of Strain BL21(DE3)-fdh

[0033] Extracting the genome of Candida boidinii as a template, and using a degenerative primer pair as primers for PCR amplification to obtain fdh gene fragments;

[0034] The nucleotide sequence of the upstream degenerate primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream degenerate primer is shown in SEQ ID NO.2:

[0035] SEQ ID NO.1: 5'-CCTCTAGAAAGGAGATATAATGAAGATYGTYTTAGTYYTWTATG-3'

[0036] SEQ ID NO.2: 5'-CCGGATCCTTTATTTCTTATCGTGTTTACCG-3'

[0037] The amplification conditions were: pre-denaturation at 94°C for 3 minutes; 30 cycles at 94°C for 30s, 58°C for 30s, and 72°C for 45s; and finally extension at 72°C for 7 minutes.

[0038] The obtained fdh gene fragment was double-digested with endonucleases XbaI and BamHI, and ligated with the plasmid pET28a(+) that was also double-digested with XbaI and BamHI (the ligation product was plasmid pET28a(+)-fdh), and transformed after 8 hours of ligation ...

Embodiment 2

[0042] Induced Expression of Formate Dehydrogenase Activity of Strain BL21(DE3)-fdh and Preparation of Crude Enzyme Solution

[0043] Inoculate BL21(DE3)-fdh into 5 mL of LB liquid medium, add 5 μL of 100 mg / mL kanamycin solution to the medium, and culture in a shaker at 37°C and 180 r / min for 12 to 24 hours . .

[0044] Transfer 3mL of bacterial liquid to 50mL of fresh LB liquid medium, and continue to cultivate at a temperature of 37°C and a shaker speed of 180r / min. After 2-3h, add IPTG solution to a final concentration of 0.5mmol / L for induction , The induction time is 3~5h.

[0045] Centrifuge to collect the bacteria completely, resuspend the bacteria with 20-30mL PBS buffer (0.1M pH7.0), then ultrasonically break (power 200-400W, condition is ultrasonic 3s, intermittent 3s, a total of 10min), centrifuge, put on The clear is the crude enzyme solution.

Embodiment 3

[0047] Determination of Formate Dehydrogenase Activity of Strain BL21(DE3)-fdh

[0048] Enzyme activity assay system: the reaction solution is 0.2mL NAD (16.7mM), 0.2mL sodium formate (1670mM) and 1.4mL PBS buffer (pH7.5, 10mM, containing 4mM DTT), add 0.2mL of diluted crude enzyme solution After mixing, place it in a spectrophotometer immediately, measure the absorbance value A1 at 340nm, react at room temperature (20°C) for 5 minutes, measure the absorbance value A2 at 340nm, A2-A1 is used to calculate the enzyme activity, and the curve of the absorbance value with time is as follows figure 2 shown.

[0049]Enzyme activity is defined as: at pH 7.5 and 30°C, the amount of enzyme required to generate 1 μmol NADH per minute is 1 enzyme activity unit (the molar extinction coefficient of NADH is 6220L·mol -1 cm -1 ).

[0050] The calculated enzyme activity is 0.6331U / mL.

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Abstract

The invention relates to a construction method and application of a formate dehydrogenase engineering strain. The construction method comprises the following steps: (1) extracting candida boidinii genome DNA as a template; (2) with the genome DNA as the template, performing PCR amplification to obtain an fdh gene segment; (3) performing double digestion on the fdh gene segment and connecting to a plasmid pET28a(+) to obtain a plasmid pET28a(+)-fdh; (4) transforming the plasmid pET28a(+)-fdh into an escherichia coli BL21(DE3) competent cell to obtain a recombinant strain BL21(DE3)-fdh for expressing fdh. According to the construction method provided by the invention, a primer pair is degenerate primers designed according to the fdh sequence of multiple strains of candida boidinii registered in NCBI (National Center of Biotechnology Information); An fdh gene can be amplified by taking the genome DNA of most candida boidinii strains as the template while the influence of individual base difference in the gene sequence is avoided.

Description

technical field [0001] The invention relates to a construction method and application of a formate dehydrogenase engineering bacterial strain, belonging to the technical field of genetic engineering. Background technique [0002] Oxidoreductases catalyze the oxidation or reduction of substrates and play a crucial role in the utilization of organic matter by organisms. Redox reactions are accompanied by the transfer of electrons, and most redox enzymes in organisms require coenzymes as electron transporters during the reaction. During the reaction, along with the oxidation of organic matter, the reduced NADH will be oxidized to its oxidized form NAD+. When NADH is consumed, the reaction will be terminated. The interconversion system of NAD+<=>NADH in organisms can continuously provide reduced NADH for oxidoreductases. When oxidoreductases are applied extracellularly, they should continuously provide reduced coenzyme NADH, while formate dehydrogenase (Formate Dehydrogen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/70A62D3/02C12R1/19A62D101/20
CPCA62D3/02A62D2101/20C12N9/0008C12Y102/01002
Inventor 张强季蕾王加宁傅晓文李天元陈贯虹
Owner ECOLOGY INST SHANDONG ACAD OF SCI
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