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Method and application for constructing PHA synthase N-terminal deletion mutant

A deletion mutant and N-terminal technology, applied in the field of molecular biology, can solve the problem of low production capacity of PHA and achieve the effect of increasing PHA production and catalytic activity

Pending Publication Date: 2021-06-29
LIAONING UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The Aeromonas hydrophila WQ (A.hydrophila WQ) PHA synthase has a wide range of substrates and produces P(3HB-co-3HHx), but its ability to produce PHA is low

Method used

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  • Method and application for constructing PHA synthase N-terminal deletion mutant
  • Method and application for constructing PHA synthase N-terminal deletion mutant
  • Method and application for constructing PHA synthase N-terminal deletion mutant

Examples

Experimental program
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Embodiment 1

[0024] Example 1 uses R.eutropha PHA synthase and Aeromonas hydrophila WQ (A.hydrophilaWQ) PHA synthase.

[0025] 1. PHA synthase gene cloning:

[0026] The sequence of Aeromonas hydrophila WQ (A.hydrophila WQ) was obtained from NCBI, and primers P1-5'-TTAAAGCTTCTAATGGAGCGCACCGCCCAG-3', P2-5'-TTTGAATTCTCATGCGGCGTCCTCCTCT-3' were designed based on this sequence. The plasmid pUC19-phaPCJ registered on NCBI WQ As a template, with P1 and P2 as primers, PCR amplification obtained phaPCJ WQ . The amplification system is: the reaction system is 50 μl: ddH 2 O3 2.5 μl; 10×Pfu Buffer 5 μl; 2.5mM dNTP 5 μl; Mg 2+ 4 μl; template DNA 1 μl; 25 μM primer 11 μl; 25 μM primer 21 μl; 5 U / μl Pfu enzyme 0.5 μl; amplification conditions: 5°C pre-denaturation for 5 min; 94°C denaturation for 30 sec; 62°C annealing for 30 sec; 72°C extension for 2.5 min, 25 Cycling; 72°C extension for 10 min.

[0027] The size of the PCR product is basically consistent with the expected result, phaC'J WQ The...

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Abstract

The invention relates to the field of molecular biology, in particular to a method for constructing a PHA synthase N-terminal deletion mutant. The method comprises the following steps that aeromonas hydrophila PHA synthase is cloned by designing a primer; and then a recombinant plasmid with deletion mutation of amino acids from the 2<nd> site to the 28 site at the N terminal is constructed. After confirming that the expression vector is successfully constructed through enzyme digestion sequencing, transforming the expression vector into an eutrophic alcaligenes mutant strain PHB-4, selecting different carbon sources, performing shake flask fermentation to verify the functional activity of PHA synthase, and then the PHA synthase is compared with wild aeromonas hydrophila. According to the method for constructing the PHA synthase N-terminal deletion mutant, the synthase gene with the mutated N end has the function of generating the PHA, and the yield of the PHA is higher than that of wild fungi, so that the yield of the PHA is increased; and a theoretical basis is provided for constructing a chimeric enzyme for producing novel PHA by using a genetic engineering technology in the future, and certain value in promoting wider application of PHA is achieved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for constructing a PHA synthase N-terminal deletion mutant. [0002] technical background [0003] Polyhydroxyalkanonate (Polyhydroxyalkanonate) is an intracellular energy and carbon source storage substance synthesized by many bacteria under nutrient imbalance conditions. PHA has good biocompatibility, biodegradability, hydrophobicity and unique properties brought by different functional groups. It is used as a tissue engineering material drug sustained release material and is a biological material with good development potential. Water-insoluble high-molecular-weight PHAs exhibit thermoplastic and / or elastic functions, as well as other physical and materialistic functions, including biodegradation, biocompatibility, piezoelectricity, etc., attracting more and more industrial attention. . PHA has many potential uses, such as commodity plastics, fishing lines, woven mat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/62C12R1/19
CPCC12N9/1025C12N15/70C12P7/625
Inventor 胡风庆李巍巍宁崇
Owner LIAONING UNIVERSITY
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