재조합 glutamate decarboxyalse를 이용한 -Aminiobutyric acid (GABA) 생산공정 최적화

Abstract

Recently, bio-based fuels and polymers from renewable biomass have drawn much attention due to the increasing environmental concerns and concern over the limited nature of fossil resources. For example, polylactic acid(PLA) made from corn has been considered as a good alternative to petroleum-based plastic and has widely been employed for general purpose plastics because of several desirable properties such as biodegradability, biocompatibility, compostability, and low toxicity to humans. However, most biopolymers cannot be applied in high-value-added areas such as automobile manufacture and electronic devices because the mechanical and thermal properties of biopolymers are not able to meet high performance requirements of such applications. Nylon 4 is four-carbon polyamide suitable for application as an engineering plastic due to its superior thermal and mechanical properties. Contrary to other nylon polymers, nylon 4 is heat-resistant, biodegradable, biocompatible and compostable. Nylon 4 is synthesized by ring-opening polymerization of petroleum-derived 2-pyrrolidone. Because 2-pyrrolidone can be prepared from gamma-aminobutyric acid(GABA), GABA produced from renewable resources such as L-glutamic acid, which is eventually used as the monomer for the synthesis of nylon 4, could broaden the industrial applications of GABA. In this study, we have demonstrated the hybrid process composed of biological and chemical processes, in which biomass-derived 2-pyrrolidone was used for the synthesis of nylon 4. High concentration of GABA was synthesized from MSG with recombinant E. coli expressing glutamate decarboxylase and then GABA was converted into 2-pyrrolidone that was finally used for the synthesis of nylon 4.

The fermentation process was developed to improve the GAD production using recombinant E. coli(BL21). The effects of ammonium sulfate, magnesium sulfate, glucose concentration, three different feeding methods and pH on the production GAD were investigated. Results showed that the production of GAD was the highest and reached 830.5 units when glucose 40 g/L, (NH4)2SO4 6 g/L, KH2PO4, 3 g/L, K2HPO4 3 g/L, yeast extract 1 g/L and 350 rpm, 7 vvm, and pH 5.5.

pH controlled batch reactor and bubble column reactors have been developed in this research. They were used to produce high concentration of GABA and to determine optimal pH for GABA production. Glutamate decarboxylase (GAD) was isolated from recombinant E. coli and used for GABA production from monosodium glutamate (MSG). pH control was inevitable because the pH increased with MSG consumption. GAD showed highest activity at acidic conditions at pH 5.5 but the optimal pH for GABA production was pH 6.0. When 1.5 mole of MSG was used as reactant, the 1.05 mole of GABA was produced after 10 hrs batch reaction. Using bubble column reactors, 80 % of MSG was converted to GABA for 6 hrs reaction and 1.2 mole of GABA was produced.

2-Pyrrolidone was synthesis with high pressure reactor using produced GABA. The sodium chloride in the GABA solution was removed using acetone precipitation to improve 2-pyrrolidone synthesis. The bulk polymerization process using 2-pyrrolidone has been developed for the production of Nylon 4. The NMR results confirmed that the produced polymer was Nylon 4.