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A kind of coenzyme regeneration system and preparation method thereof

A technology of coenzyme regeneration and enzyme solution, applied in the field of coenzyme regeneration system and preparation thereof, can solve the problems of high cost, difficult to reuse, poor stability, etc., and achieve the effects of high coenzyme regeneration efficiency, simple and convenient preparation method, and cost reduction.

Active Publication Date: 2019-04-09
ABA CHEM CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These coenzymes are often more expensive than the prepared products, have poor stability and are difficult to reuse, resulting in high reaction costs in industrial production, thus limiting the use of oxidoreductases

Method used

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  • A kind of coenzyme regeneration system and preparation method thereof
  • A kind of coenzyme regeneration system and preparation method thereof
  • A kind of coenzyme regeneration system and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: PCR amplification target gene

[0056] (1) PCR amplification primers

[0057] Primers were designed according to the gene sequence of formate dehydrogenase (FDH) threonine deaminase (TD) (Genbank number: XM_001525495 and AAB18593) and its sequence. After screening and verification, the best upstream primer for formate dehydrogenase (FDH) is 5'-AAA CATATG AAAATCGTTCTCGTTTTGTACTCC-3' (the underline is the NdeI restriction site); the downstream primer is 5'-AAA CTCGAG TGCGACCTTTTTGTCATTAC-3' (the underline is the XhoI restriction site). The upstream and downstream primers were designed according to the gene sequence of threonine deaminase (AAB18593). After screening and verification, the best upstream primer was 5'-AAA AAGCTT GTTAGCAGCCGGATCTCAG-3' (the underline is the HindIII restriction site); the downstream primer is 5'-AAA CATATGGTATTAAAACAAATTCTTC-3' (the underline is the NdeI restriction site).

[0058] Using the total DNA of Candida and Esche...

Embodiment 2

[0068] Embodiment 2: Construction of recombinant plasmid

[0069] The plasmid construction process is as follows figure 1 As shown (taking the construction of FDH as an example), using the total Candida DNA as a template, use the specific endonucleases NdeI and XhoI to double-digest the amplified gene fragment or the gene fragment connected to the T vector, and double-digest at the same time After the vector plasmid was purified and recovered, it was ligated at 16°C for 5 hours, and the ligated product was transformed into a competent cell E.coli DH5α. A single colony transformed on an LB solid plate was picked and transferred to 5 mL of LB liquid medium (according to Add the corresponding antibiotics for the resistance of the plasmid), and culture overnight at 37°C in a shaker at 220r / min. The plasmid was extracted, and the plasmid was picked for double enzyme digestion verification. After the verification was correct, the constructed plasmid pFDH-pET28a was subjected to DNA...

Embodiment 3

[0076] Example 3: Construction of co-expressed recombinant strains

[0077] Co-transform pFDH-pET28a and pLDH-pET21a into E.coli BL21(DE3), and spread on LB (Amp and Kan each 100μg / ml) solid plate, on LB (Amp and Kan each 100μg / ml) solid plate The single bacterium colony that grows on above is exactly the bacterial strain that simultaneously contains formate dehydrogenase and leucine dehydrogenase gene, will obtain genetically engineered bacterial strain and name E.coliBL21-FDH / LDH. The strain can simultaneously express formate dehydrogenase and leucine dehydrogenase.

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Abstract

Provided is a coenzyme regeneration system, comprising: a formate dehydrogenase (FDH), a leucine dehydrogenase (LDH) and an ammonium formate. Also provided is a method for preparing the coenzyme regeneration system, comprising the steps of: (1) heterogeneous expression of the formate dehydrogenase and the leucine dehydrogenase; and (2) preparation of the coenzyme regeneration system.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a coenzyme regeneration system and a preparation method thereof. Background technique [0002] Compared with traditional chemical catalysis, biocatalysis has attracted much attention and research because of its high chemoselectivity, regioselectivity and stereoselectivity of enzyme-catalyzed reactions. Among them, due to the high efficiency and specificity of oxidoreductases, they occupy a very important position in biosynthesis. Oxidoreductases can selectively catalyze compounds containing carbonyl groups, aldehydes and ketones. Oxidoreductase is used as a catalyst to prepare chiral compounds, and a certain amount of coenzyme will be consumed while synthesizing the product. For example: Li Shuting, etc. prepare L-tert-leucine with trimethylpyruvate, use leucinase and formate dehydrogenase coupling system in the synthesis, formate dehydrogenase can be regenerated by coenzyme I, and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/00C12N15/70C12N15/66
CPCC12P1/00
Inventor 李航徐军阙利民江岳恒蔡彤
Owner ABA CHEM CORP
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