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A kind of method and application of in vitro enzymatic reaction to generate 1,2,4-butanetriol

A butanetriol and in vitro enzyme technology, which is applied in the field of bioengineering to achieve the effects of shortening production cycle, increasing product quantity and increasing activity

Active Publication Date: 2018-02-02
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the synthesis of 1,2,4-butanetriol by using D-xylonic acid as a substrate in vitro.

Method used

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  • A kind of method and application of in vitro enzymatic reaction to generate 1,2,4-butanetriol
  • A kind of method and application of in vitro enzymatic reaction to generate 1,2,4-butanetriol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Preparation of crude enzyme solution of enzymes related to 1,2,4-butanetriol anabolic metabolism, the specific steps are as follows:

[0038] (1) Use primers at the 5' end and 3' end of the yjhG gene to introduce restriction sites (NcoI and EcoRI), carry out double enzyme digestion on the yjhG gene and pET-30a(+), and then connect the yjhG gene to pET-30a ( +) on the carrier;

[0039] (2) Use primers at the 5' end and 3' end of the mdlC gene to introduce restriction sites (NcoI and EcoRI), carry out double enzyme digestion on the mdlC gene and pET-30a(+), and then connect the mdlC gene to pET-30a ( +) on the carrier;

[0040] (3) Utilize adhP gene 5' and 3' end primers to introduce restriction sites (NcoI and EcoRI), carry out double enzyme digestion to adhP gene and pET-30a (+), then connect adhP gene to pET-30a ( +) on the carrier;

[0041] (4) Transform the corresponding three recombinant plasmids constructed in steps (1), (2), and (3) into Escherichia coli BL21 (...

Embodiment 2

[0044] The in vitro reaction of enzymes related to 1,2,4-butanetriol synthesis and metabolism, the specific steps are as follows:

[0045] (1) Take 100ml of E. coli culture solution expressing the above three enzymes at 4200rpm, centrifuge at 4°C for 5min, discard the supernatant, wash twice with 50mM pH 7.0 Hepesbuffer, and finally resuspend with 5ml Hepesbuffer; then break it with a sonicator (22kHz, 150w), crush for 30min, centrifuge the crushed solution at 15000rpm at 4°C for 10min, separate the supernatant from the precipitate, keep the supernatant, add 10% glycerol, and measure the protein content according to the kit method.

[0046] (2) The D-xylonate dehydratase, 2-ketoacid decarboxylase, and alcohol dehydrogenase prepared in step (1) are added to the reaction system, and the concentration of D-xylonate dehydratase is kept at 0.39 mg / ml, 2- The concentration of ketoacid decarboxylase is 0.05mg / ml, the concentration of alcohol dehydrogenase is 0.39mg / ml, and other reac...

Embodiment 3

[0049] Intermediate level 1,2,4-butanetriol is synthesized in vitro, and the specific steps are as follows:

[0050] (1) Take 100ml of E. coli culture solution expressing the above three enzymes at 4200rpm, centrifuge at 4°C for 5min, discard the supernatant, wash twice with 50mM pH 7.0 Hepes buffer, and finally resuspend with 5ml Hepes buffer; Instrument crushing (22kHz, 150w), crushing 30min, crushing liquid 15000rpm 4 ℃ centrifugation 10min, supernatant and precipitate were separated, supernatant was retained, 10% glycerol was added, and the protein content was determined according to the kit method.

[0051] (2) The D-xylonate dehydratase, 2-ketoacid decarboxylase, and alcohol dehydrogenase prepared in step (1) are added to the reaction system, and the concentration of D-xylonate dehydratase is kept at 0.39 mg / ml, 2- The concentration of ketoacid decarboxylase is 0.5mg / ml, the concentration of alcohol dehydrogenase is 0.39mg / ml, and other reaction substances are added, the...

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Abstract

The invention discloses a method of generating 1,2,4-butantriol by the in vitro enzyme reaction and application thereof, and belongs to the technical field of bioengineering. The method provided by the invention comprises the following steps: respectively constructing genetically-engineered bacteria of over-expressed D-xylonic acid anhydrase genes, 2-keto acid decarboxylase genes and alcohol dehydrogenase genes; after carrying out fermentation cultivation on the obtained genetically-engineered bacteria, crushing thalli by ultraviolet waves; collecting crude enzyme fluid; and after carrying out mixed adjustment on concentration of D-xylonic acid anhydrase, 2-keto acid decarboxylase and alcohol dehydrogenase, adding a reaction substrate to synthesize 1,2,4-butantriol. The method provided by the invention realizes synthesis of 1,2,4-butantriol in vitro by controlling the enzyme reaction and using D-xylonic acid as the raw material and has the characteristic of good convenience for enlarging production; and the enlarged yield can reach 5.98g / L.

Description

technical field [0001] The invention relates to a method and application for generating 1,2,4-butanetriol by in vitro enzyme reaction, belonging to the technical field of bioengineering. Background technique [0002] 1,2,4-Butanetriol (BT) is a colorless, odorless, transparent water-soluble viscous syrupy polyol, which is mainly used as a chemical intermediate in organic synthesis, widely used in military industry, medicine, Tobacco, cosmetics, paper, agriculture and polymer materials fields. Its nitro compound 1,2,4-butanetriol trinitrate is a high-energy plasticizer with civil and military potential, and it can replace nitroglycerin as a high-energy, high-tech propellant. In medicine, BT can be used as a sustained-release agent to control the release rate of drugs. It is a key intermediate for the synthesis of antiviral compounds, platelet activating factors and other drugs. In the field of polymer materials, it can be used as a cross-linking agent for polymer materials;...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/18C12N9/88C12N9/04C12N15/70
CPCC12N9/0006C12N9/88C12P7/18C12Y401/01001C12Y402/01082
Inventor 咸漠蒋昱东刘炜曹玉锦
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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