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Preparation method of lactate polymers and lactate copolymers using polyhydroxyalkanoate synthase mutants

A technology of polyhydroxyalkanoate and lactate, applied in the field of mutants, can solve the problems of low industrial efficiency and difficulty in synthesizing PLA copolymers

Inactive Publication Date: 2012-07-11
LG CHEM LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] When PLA is synthesized from lactate by chemical synthesis, PLA homopolymers can be easily produced, however, the synthesis of PLA copolymers with various monomer compositions is difficult and has very low industrial efficiency

Method used

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  • Preparation method of lactate polymers and lactate copolymers using polyhydroxyalkanoate synthase mutants
  • Preparation method of lactate polymers and lactate copolymers using polyhydroxyalkanoate synthase mutants
  • Preparation method of lactate polymers and lactate copolymers using polyhydroxyalkanoate synthase mutants

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Embodiment 1

[0063] Gene cloning and research of PHA synthase having high homology to the amino acid sequence of PHA synthase derived from Pseudomonas bacterium MBEL 6-19

[0064] In order to study the polyhydroxyalkanoate synthase (PhaC1) derived from Pseudomonas MBEL 6-19 (KCTC 11027BP) used in the present invention Ps6-19 ) a polyhydroxyalkanoate synthase with a high degree of amino acid sequence homology was analyzed using the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information (NCBI), and in the results Synthases showing relatively high amino acid sequence homology are shown in Table 1. All enzymes are included in the group of type II polyhydroxyalkanoate synthases, which are medium chain length (MCL) enzymes used to polymerize substrates with relatively long carbon chains. - PHA synthase.

[0065] [Table 1] Shown with PhaC1 Ps6-19 Polyhydroxyalkanoate synthase with high amino acid sequence homology

[0066]

[0067]

[00...

Embodiment 2

[0093] Preparation of mutants produced by highly active lactate polymers and copolymers

[0094] Type II polyhydroxyalkanoate synthases are considered to be MCL-PHA synthases that polymerize substrates with relatively long carbon chains, and it has been reported that the synthesis of short chain length (SCL)-PHA by studying various mutations Studies on mutants with improved activity (WO08 / 062999; Takase et al., J.Biochem., 2003, 133: 139-145; Takase et al., Biomacromolecules, 2004, 5: 480-485; Matsumoto et al., 2005, Biomacromolecules, 6: 99-104; Matsumoto et al., 2006, Biomacromolecules, 7: 2436-2442).

[0095] For the present invention, the mutants shown in Table 3 below were prepared by using the SDM method with primers of SEQ ID NOS: 27 to 74 to affect the lactate polymer and Amino acid sequence mutations for the synthetic activity of copolymers were introduced into four newly produced type II polyhydroxyalkanoate synthases (PhaC1 Pch , PhaC1 Ppu , PhaC1 Pre and PhaC1...

Embodiment 3

[0150] using synthase mutants to produce lactate polymers and copolymers

[0151] In order to quantitatively analyze five kinds of wild-type type II polyhydroxyalkanoate synthases (PhaC1) prepared in Example 2 Ps6-19 , PhaC1 Pch , PhaC1 Ppu , PhaC1 Pre and PhaC1 Pae ) and its mutant lactate copolymer [P(3HB-co-LA)], at 30°C in MR medium supplemented with glucose (20g / L) and 3HB (2g / L) E. coli XL-1 Blue transformed with the recombinant expression vector (Table 3) containing the above-mentioned synthase was cultured in the flask for 4 days. The composition of the MR medium used in the present invention is listed in Table 4. The cultured bacteria were collected by centrifugation, washed three times with distilled water, and then dried in an oven at 100°C for 24 hours. The composition and content of the polymers synthesized in the stem cells were analyzed by gas chromatography, and the results are shown in Table 5.

[0152] [Table 4] Composition of MR medium for cultivatio...

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Abstract

The present invention relates to diverse polyhydroxyalkanoate synthase (PHA synthase) mutants capable of synthesizing lactate polymers (PLAs) and lactate copolymers (PLA copolymers), and a preparation method of the lactate polymers and copolymers using the PHA synthase mutants. More specifically, the present invention relates to polyhydroxyalkanoate synthase mutants having SEQ. ID. NO: 2, 4, 6 or 8; and a preparation method of PLA polymers and PLA copolymers using the synthase mutants. According to the present invention, polyhydroxyalkanoate synthase mutants having SEQ. ID. NO: 2, 4, 6 or 8 can have PLA polymer and copolymer synthase activity by the variation in amino acid sequence that affects the PLA polymer synthase activity. Further, the use of such synthase mutants makes it possible to prepare PLA polymers and copolymers having different properties from each other.

Description

technical field [0001] The present invention relates to mutants of various polyhydroxyalkanoate synthases capable of synthesizing lactate polymers and lactate copolymers using lactyl-CoA (lactyl-CoA) as a substrate. Moreover, the present invention also relates to a method for preparing lactate polymers and lactate copolymers using a mutant of polyhydroxyalkanoate synthase having increased The activity of synthetic lactate polymers and lactate copolymers. Background technique [0002] Polylactate (PLA) is a common biodegradable polymer derived from lactate, which is very suitable as a polymer for medical use or general use. Currently, PLA is produced by the polymerization of lactic acid esters produced by microbial fermentation, however, this direct polymerization of lactic acid esters can only produce PLA with low molecular weight (1000-5000 Daltons). There is a method of polymerizing low-molecular-weight PLA produced by direct polymerization of lactate using a chain coupl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12P7/56
CPCC12P7/56C12Y208/03001C12P7/625C12N9/13C12N9/00C12N15/52
Inventor 梁宅镐姜惠玉李相烨郑有景
Owner LG CHEM LTD
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