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Method for producing isoprene by in vitro enzymatic reaction and application of isoprene

A kind of isoprene, in vitro enzyme technology, applied in the field of bioengineering

Inactive Publication Date: 2015-04-01
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 construction of isoprene synthetic metabolic pathways in vitro using mevalonate as a substrate using five crude proteins

Method used

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  • Method for producing isoprene by in vitro enzymatic reaction and application of isoprene
  • Method for producing isoprene by in vitro enzymatic reaction and application of isoprene

Examples

Experimental program
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Effect test

Embodiment 1

[0047] (1) Take 100ml of E. coli culture solution expressing the above five enzymes at 7000rpm, centrifuge at 4°C for 5min, discard the supernatant, wash twice with 50mM, pH 7.4 phosphate buffer, and finally use 5ml phosphate buffer to resuspend; then use Ultrasonic crusher (22kHz, 150w), after crushing, centrifuge at 15000rpm at 4°C for 5min, separate the supernatant from the precipitate, keep the supernatant, add 10% glycerol, and measure the protein content according to the kit method.

[0048] (2) In order to investigate the action strength of each enzyme in the catalytic reaction, the obtained phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl pyrophosphate isomerase, isopentyl The concentration of diene synthase was fixed at 120 μg / L, and only the amount of mevalonase was changed. 160μg / L, do six reactions, the final reaction system is: 50mM phosphate buffer, 4mM ATP, 30mM KCl, 8mM MgCl2, 0.2mM MnCl2, 0.01mM ZnSO4, 4mM β-mercaptoethanol, 2.5mM ...

Embodiment 2

[0051] (1) Take 100ml of E. coli culture solution expressing the above five enzymes at 7000rpm, centrifuge at 4°C for 5min, discard the supernatant, wash twice with 50mM, pH 7.4 phosphate buffer, and finally use 5ml phosphate buffer to resuspend; then use Ultrasonic crusher (22kHz, 150w), after crushing, centrifuge at 15000rpm at 4°C for 5min, separate the supernatant from the precipitate, keep the supernatant, add 10% glycerol, and measure the protein content according to the kit method.

[0052] (2) In order to investigate the action strength of each enzyme in the catalytic reaction, the mevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl pyrophosphate isomerase, and isopentyl diphosphate obtained in step (1) were The concentration of ene synthase was fixed at 120 μg / L, and only the amount of phosphomevalonase was changed, the concentrations were 0 μg / L, 62.5 μg / L, 80 μg / L, 100 μg / L, 120 μg / L, 140 μg / L, 160μg / L, do six reactions, the final reaction system ...

Embodiment 3

[0055] (1) Take 100ml of E. coli culture solution expressing the above five enzymes at 7000rpm, centrifuge at 4°C for 5min, discard the supernatant, wash twice with 50mM, pH 7.4 phosphate buffer, and finally use 5ml phosphate buffer to resuspend; then use Ultrasonic crusher (22kHz, 150w), after crushing, centrifuge at 15000rpm at 4°C for 5min, separate the supernatant from the precipitate, keep the supernatant, add 10% glycerol, and measure the protein content according to the kit method.

[0056] (2) In order to investigate the action strength of each enzyme in the catalytic reaction, the mevalonate kinase, phosphomevalonate kinase, isopentenyl pyrophosphate isomerase and isoprene obtained in step (1) were synthesized The enzyme concentration was fixed at 120 μg / L, and only the amount of mevalonate pyrophosphate decarboxylase was changed. 160μg / L, do six reactions, the final reaction system is: 50mM phosphate buffer, 4mM ATP, 30mM KCl, 8mM MgCl2, 0.2mM MnCl2, 0.01mM ZnSO4, 4m...

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Abstract

The invention discloses a method for producing isoprene by in vitro enzymatic reaction and an application of isoprene and belongs to the technical field of biological engineering. The method disclosed by the invention comprises the following steps of respectively introducing an obtained mevalonate kinase gene, a phosphomevalonate kinase gene, a mevalonate-pyrophosphate decarboxylase gene, an isopentenyl pyrophosphate isomerase gene and an isoprene synthase into host bacteria, carrying out fermental cultivation to obtain five recombinant bacteria, respectively carrying out ultrasonic disruption on each thallus to obtain a crude enzyme solution, mixing five crude enzyme solutions to obtain a reaction enzyme solution and adding a reaction substrate solution into the reaction enzyme solution to in vitro synthesize isoprene. By the method disclosed by the invention, the in-vitro synthesis of isoprene from mevalonic acid by virtue of enzymatic reaction is achieved and the synthesis of isoprene by precisely in vitro controlling the content of metabolic enzyme is realized.

Description

technical field [0001] The invention relates to a method for generating isoprene by in vitro enzyme reaction and its application, belonging to the technical field of bioengineering. Background technique [0002] Isoprene is an important chemical platform compound, 95% of which is used in synthetic rubber; it is also the second monomer of butyl rubber. In addition, isoprene is also widely used in the fields of pesticides, medicines, spices and adhesives. At present, the source of isoprene is mainly through petroleum-based raw material isopentane, isopentene dehydrogenation method, chemical synthesis method (including isobutylene-formaldehyde method, acetylene-acetone method, propylene dimerization method) and cracking C5 fraction extraction Distillation. However, with the increasing depletion of fossil resources, the source of raw materials is an important bottleneck issue for the preparation of isoprene from petroleum-based raw materials. [0003] With the development of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P5/02C12R1/19C12R1/125C12R1/685
Inventor 咸漠程涛赵广邹慧斌
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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