Trifunctional enzyme for de novo synthesis of flavanone as well as synthesis method and application of trifunctional enzyme

A technology of trifunctional enzyme and synthesis method, which is applied in chemical instruments and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of low yield of in vitro enzymatic synthesis, difficult formation of substrate channels, cumbersome production process, etc. Overcome the effects of low yield and substrate conversion, considerable economic benefits, and easy operation

Active Publication Date: 2021-10-22
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, as far as the in vitro enzymatic synthesis of complex compounds is concerned, the actual production process is cumb

Method used

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  • Trifunctional enzyme for de novo synthesis of flavanone as well as synthesis method and application of trifunctional enzyme
  • Trifunctional enzyme for de novo synthesis of flavanone as well as synthesis method and application of trifunctional enzyme
  • Trifunctional enzyme for de novo synthesis of flavanone as well as synthesis method and application of trifunctional enzyme

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Embodiment 1

[0036] 1. Cloning, expression and purification of key enzymes in the flavanone biosynthetic pathway

[0037] Cloning of 4-coumaroyl-CoA ligase (4CL) gene from soybean by conventional method 4CL1 , Cloning of chalcone synthase (CHS) gene from sorghum CHS2 To the prokaryotic expression vector pET-32a(+), construct the recombinant plasmid pET-32a- 4CL1 and pET-32a- CHS2 . Then the recombinant plasmid pET-32a- 4CL1 and pET-32a- CHS2 Transform Escherichia coli BL21(DE3) respectively, induce gene expression by 0.1 mM IPTG at 20°C for 3-4 hours, and use bacterial lysis buffer (50 mM NaH 2 PO4, 300 mM NaCl, pH8.0, 1 mM DTT, 1 mM PMSF, 1 μg / mL aprotinin, 1 μg / mL leupeptin and 1 μg / mL pepsin) After sonicating the bacterial suspension, Use nickel agarose gel to purify the recombinant protein according to the manufacturer's instructions (BeyoGold His-tag Purification Resin reagent instructions), add 1 mM DTT, concentrate the protein in the ultrafiltration centrifuge tube to obtai...

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Abstract

The invention discloses a trifunctional enzyme for de novo synthesis of flavanone, wherein 4-coumaroyl-coenzyme A ligase genes, chalcone isomerase genes and chalcone synthase genes are cloned to a prokaryotic expression vector to construct recombinant plasmids, then escherichia coli is converted to induce expression of recombinase; and then purified recombinase is utilized to establish a cell-free synthesis system, and in-vitro enzymatic synthesis of flavanone is realized. According to the invention, the problems of low yield and low substrate conversion rate in an in-vitro multi-enzyme synthesis system of flavanone are solved, and an in-vitro enzymatic synthesis system with simple components is established at the same time. The system does not have a complex regulation effect in engineering bacteria cells, the reaction process is easy to accurately control, byproducts are few, product separation is relatively simple, the production period is obviously shortened, and the production cost is obviously reduced.

Description

technical field [0001] The invention relates to a trifunctional enzyme for de novo synthesis of flavanones, its synthesis method and application, the classification number is C12P, and it belongs to the technical field of biomedicine. Background technique [0002] Flavonoids generally refer to a large class of natural small molecule plant secondary metabolites whose basic core is 2-phenylchromone, and now generally refer to two benzene rings connected to each other through the central three-carbon chain to form a C6-C3-C6 A series of compounds with the main structure. According to the difference of the central C ring, flavonoids are generally divided into six subgroups: flavanols, flavonoids, flavonols, flavanones, anthocyanins and isoflavones. These compounds have a variety of biological and pharmacological activities, and are currently one of the hot spots in the field of biomedicine, and have broad application and development prospects in medicine, food, cosmetics and ot...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N9/90C12N9/10C12N15/70C12P17/06
CPCC12N15/52C12N9/93C12N9/90C12N9/1037C12N15/70C12P17/06C12Y203/01074C12Y602/01012C12Y505/01006C07K2319/00C12N2800/22Y02A50/30
Inventor 张新跃何妍之聂也森张智萍丁笠陈磊廖凯赵晨宏
Owner YANGZHOU UNIV
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