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Enol dehydrogenase, encoding gene, carrier, engineering bacteria and application of enol dehydrogenase

A technology of enol dehydrogenase and encoding gene, which is applied in the field of enol dehydrogenase and its encoding gene, can solve the problems of reducing the yield of target reaction products, side reactions, etc.

Active Publication Date: 2014-03-05
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the cell contains a variety of enzymes, the whole cell catalysis often encounters the problem of side reactions, and the existence of side reactions will reduce the yield of the target reaction product

Method used

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  • Enol dehydrogenase, encoding gene, carrier, engineering bacteria and application of enol dehydrogenase
  • Enol dehydrogenase, encoding gene, carrier, engineering bacteria and application of enol dehydrogenase
  • Enol dehydrogenase, encoding gene, carrier, engineering bacteria and application of enol dehydrogenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Use the nucleic acid extraction kit to extract the genomic DNA of Yorkella sp.WZY002 (Yokenella sp. AGCTATGCCGCAAAAGAG-3'), primer 2 (5'-AAAGTCGGCTTGCAGTACC-3') for PCR amplification.

[0065] The amount of each component in the PCR reaction system (total volume 50 μL):

[0066] 10×TransStart rTaq Buffer (with Mg 2+ ) 5 μL, 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP and dTTP) 12.5 μL, 1 μL each of primer 1 (1-1 or 1-2) and primer 2 at a concentration of 50 pM, 1 μL of genomic DNA, 1 μL of TransTaq DNA polymerase , add water to make up to 50 μL.

[0067] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 4min, followed by a temperature cycle at 94°C for 30s; 55°C for 30s; 72°C for 1min; a total of 30 cycles, and a final extension at 72°C for 10min with a termination temperature of 4°C.

[0068] Take 6 μL of the PCR reaction solution and use 0.8% agarose gel electrophoresis to detect it. The electrophoresis result shows that it is a sing...

Embodiment 2

[0070]The recombinant plasmid obtained according to Example 1 was chemically transformed into Escherichia coli Trans B (DE3) (the competent cells were purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and the recombinant plasmid pEASY-E2-YsADH containing intracellular expression was obtained Recombinant Escherichia coli Trans B(DE3) / pEASY-E2-YsADH. The recombinant Escherichia coli was cultured with LB liquid medium containing ampicillin (50 μg / ml) and kanamycin (50 μg / ml) at 37°C for 16 hours, and then inoculated with 1% inoculum (v / v) into fresh containing Ampicillin (50 μg / ml) and kanamycin (50 μg / ml) in LB liquid medium, cultured at 37°C until the cell concentration OD600 is about 0.6, and then added to the LB liquid medium with a final concentration of 0.2mM IPTG, induced at 22°C for 6-8 hours, then centrifuged at 10,000 rpm for 10 minutes at 4°C, and collected bacterial cells containing recombinant enol dehydrogenase. The obtained recombinant genetically engine...

Embodiment 3

[0075] The recombinant Escherichia coli Trans B (DE3) / pEASY-E2-YsADH wet cell was obtained in Example 2, and the cell was resuspended with 50mM Tris-HCl buffer solution of pH 8.0, and then ultrasonicated (sonication 2s, interval 6s , the effective ultrasonic time is 5min), the broken suspension is centrifuged at 10000rpm at 4°C for 10min, and the cell debris is discarded as much as possible to obtain the protein crude enzyme solution. According to the instructions of Ni-NTA metal chelate affinity chromatography, take the protein crude enzyme solution and load it on the pre-equilibrated Ni 2+ In the column, use 10mM imidazole, 40mM imidazole, 100mM imidazole, 250mM imidazole to elute the impurity protein and target protein in sequence. After the recombinant enol dehydrogenase was eluted with 100 mM imidazole, the obtained enzyme solution was desalted and concentrated by ultrafiltration membrane, and then stored at -20°C.

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Abstract

The invention provides an enol dehydrogenase and an encoding gene thereof, a carrier containing the encoding gene, engineering bacteria and application of the enol dehydrogenase. An amino acid sequence of the enol dehydrogenase is shown in SEQ ID No.1; the encoding gene is shown in SEQ ID No.2. The invention provides a nucleotide sequence of an enol dehydrogenase gene from Yokenella WZY002 (Yokenella sp.WZY002). The enol dehydrogenase gene can be connected with an expression carrier to build an intracellular expression recombinant plasmid containing the gene, and the intracellular expression recombinant plasmid is transformed into an escherichia coli strain, so as to obtain recombinant escherichia coli. The recombinant escherichia coli is subjected to cell disruption, separation and purification, so as to obtain the recombinant enol dehydrogenase. The recombinant enol dehydrogenase has the ability of reducing substrates, such as olefine aldehyde, olefine ketone, aromatic aldehyde, aromatic ketone and the like to generate corresponding alcohol. Oxidation of alcohol and reduction of aldehyde are simultaneously catalyzed by the recombinant escherichia coli or the recombinant enol dehydrogenase as a biological catalyst, and crotonaldehyde, benzyl alcohol, nerol and geraniol can be generated.

Description

(1) Technical field [0001] The invention relates to an enol dehydrogenase and its encoding gene, a carrier containing the encoding gene, engineering bacteria and application thereof. (2) Background technology [0002] α,β-Unsaturated compounds such as enols, enaldehydes and enones have important applications in many fields of the chemical industry, and are often used in the production of fine chemicals, drugs and flavors and fragrances. Currently, α,β-unsaturated enols are usually prepared by selective hydrogenation of enals or enones using chemical catalysts. The chemical reduction of α,β-unsaturated alkenes or enones to prepare enols often has low selectivity and is accompanied by the formation of saturated alcohols as by-products. As an alternative to chemical methods, biocatalytic preparation of α,β-unsaturated enols has the advantages of high efficiency, economy, high selectivity, and mild conditions, and has been paid more and more attention by researchers. Unlike ch...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/24C12P7/22C12P7/04C12R1/01
CPCC12N9/0006C12P7/04C12P7/22C12P7/24
Inventor 应向贤汪钊王一芳郑建永章银军
Owner ZHEJIANG UNIV OF TECH
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