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Polyhydroxyalkanoate in-vitro synthesis method

A polyhydroxyalkanoate, in vitro synthesis technology, applied in the direction of fermentation, etc., to achieve the effect of controllability

Active Publication Date: 2019-06-21
CHANGCHUN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to solve the problem in the prior art that PHA can only be prepared by fermentation based on the metabolic pathway of microorganisms, and then provide an in vitro synthesis method that does not rely on the metabolic pathway of microorganisms

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preparation example Construction

[0029] The polyhydroxy fatty acid copolyester P(3HB-co-2HB) of the present invention is synthesized in vitro by microorganisms, and the raw materials for in vitro synthesis mainly include the synthesis of the substrate (R)-3HB and the related enzyme ACS in the synthesis pathway of (R)-2HB Enzyme (hereinafter referred to as ACS), PCT synthetase (hereinafter referred to as PCT), ReC synthetase (hereinafter referred to as ReC) and auxiliary reagents required for the reaction. The synthetic method mainly comprises the following steps:

[0030] The ACS synthetase, PCT synthetase and ReC synthetase (ACS0.2mg / mL, PCT0.2mg / mL, ReC0.5mg / mL) obtained by in vitro recombinant expression. Add to the aqueous phase system containing 200mM (R)-3HB and (R)-2HB and mix (the aqueous phase system solution includes: 100mM sodium dihydrogen phosphate, 10mM magnesium chloride, 30mM ATP, 0.2mg / ml bovine serum albumin, 1mM CoA and 10mM acetic acid), at 28-31°C for 60-84h to react.

[0031] According...

specific Embodiment approach 1

[0042] The in vitro synthesis of polyhydroxy fatty acid copolyester P (3HB-co-2HB) in this embodiment comprises the following steps:

[0043]Step 1. Construction of expression strains: the pCold-acs, pQE-pct, and pCold-phaC plasmids were introduced into Escherichia coli BL21 (DE3) by thermal transformation, and the transformants were spread on the LB-Amp solid culture plate, 37 Cultivate overnight. Pick the single clone grown on the plate, inoculate it into LB-Amp liquid medium, culture it on a shaker at 37°C and 180rpm for 8 hours, extract the plasmid and perform gene sequencing verification, and the obtained sequences are all correct. The BL21 (DE3) recombinant bacteria containing pCold-acs were named BL21-ACS; the BL21 (DE3) recombinant bacteria containing pQE-pct were named BL21-PCT; the BL21 (DE3) recombinant bacteria containing pCold-phaC were named BL21-ReC.

[0044] Step 2. Induced expression of target protein: inoculate BL21-ACS, BL21-PCT, and BL21-ReC overnight act...

specific Embodiment approach 2

[0056] Specific embodiment 2: The difference from specific embodiment 1 is that 3HB is referred to as 2HB in the addition ratio: 3HB is added in a volume of 750 μL, 2HB is added in a volume of 250 μL, and the rest of the components are added according to Table 1. After the reaction, 4.7 mg of polymer is obtained. Hydroxy fatty acid esters. Among them, the ratio of 2HB / 3HB is 25 / 75.

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Abstract

The invention relates to a polyhydroxyalkanoate in-vitro synthesis method. The method includes: taking (R)-3-hydroxybutyric acid (3HB) and (R)-2-hydroxybutyric acid (2HB) as a substrate, adding related substrate precursor synthetases including acetyl coenzyme A synthetase (ACS), fatty acid coenzyme A transferase (PCT) and substrate precursor polymerase PHA synthase (ReC) in a designed substrate polymerization way into a water-phase synthesis system, standing for reaction at 28-31 DEG C for 60-84h to synthesize copolyester P (3HB-co-2HB). According to the method, PHA synthesis related enzymes obtained through gene in-vitro overlap expression serve as catalysts, substances which cannot be used by microorganisms or toxic to the microorganisms can be used as substances, and an environment-friendly biomaterial with novel material properties can be developed. On the basis of advantages of substrate proportion adjustability and reaction process visualization of the synthesis method, adjustability of content of any components for copolyester synthesis and intelligent detection of a whole reaction process can be realized.

Description

technical field [0001] The invention relates to the field of polyhydroxyalkanoate (PHA) synthesis, in particular to an in vitro synthesis method of polyhydroxyalkanoate copolyester. Background technique [0002] Polyhydroxyalkanoate (PHA) is an intracellular polyester substance that can be produced by a variety of microorganisms. It is mainly a substance produced by microorganisms in cells and used as an energy reserve when the culture conditions are unbalanced (such as sufficient nitrogen source or insufficient carbon source). Because PHA has the same thermoplasticity as petroleum-based plastics and also has biodegradability that petroleum-based plastics do not have, PHA is a new environmentally friendly biomaterial that replaces traditional petroleum-based plastics. [0003] PHA is divided into two types according to the number of carbon atoms: short-chain (SCL) PHA, whose monomers are composed of 3-5 carbon atoms; medium and long-chain (MCL) PHA, whose monomers are compo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/62
Inventor 韩雪容徐飞宏
Owner CHANGCHUN UNIV OF SCI & TECH
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