Primary recovery of Gamma-aminobutyric acid from cell culture using the aqueous two-phase system

• Main Authors: Sona Jabang, 蘇娜
• Other Authors: Chi-Wei Lan
• Format: Others
• Language: en_US
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/7xk7k7

碩士 === 元智大學 === 生物科技與工程研究所 === 106 === Aqueous biphasic system, liquid-liquid fractionating technique, is usually formed as a result of mutual incompatibility of two polymers, solvents or one polymer and a salt above a critical concentration. Owing to its great potentials in the extraction, separation, purification, enrichment of proteins, viruses, enzymes, nucleic acid and other biomolecules made it extensively used in both industry and academic field. Hence, this simple and scalable technique was employed in the present study to recover a valuable compound, Gamma-aminobutyric acid (GABA). The GABA, a non-proteinous amino acid, is the principal inhibitory neurotransmitter in the central nervous system of mammalian. It is ubiquitous in vertebrates, and in microbes and insects as a metabolite. The physiological functions of GABA have been felt on victims suffering from tranquillizers, anxiety, depression, Alzheimer, Schizophrenia, diabetes and hormonal imbalance, medically. Pharmaceutically, drugs are invented to enhance the availability of GABA. Besides, it serves as the major building block for the synthesis of 2-pyrrolidone and biodegradable polyamide nylon 4 making it boost the interest of the industrial field. GABA is biologically synthesized from the principal excitatory neurotransmitter, L glutamate, by glutamate decarboxylase in the presence of pyridoxal 5’ phosphate. The increasing demand of GABA has prompted scientist to elucidate the efficient production of GABA. However, separation remains in the challenging manner. To this end, polyethylene glycol of different molecular weight (2000, 4000, 6000 and 8000) /dipotassium hydrogen phosphate and potassium dihydrogen phosphate (K2HPO4 and KH2PO4) at different physiological pH was utilized for the construction of binodal curve and tie lines. In the case of alcohol-salt base, ethanol/methanol and dipotassium hydrogen phosphate/tri sodium citrate and ammonium sulfate were used for the phase diagram construction. Separation of GABA was done using High-Performance Liquid Chromatography. The derivatization was completed with 1, fluoro 2,4 dinitrobenzene (FDNB). During the phase formation, methanol/tri sodium citrate could not form two phases due to the heavy salting out effect. Although, salting out effect was observed in ethanol/ammonium phase formation, yet phase formation was successful. Equally, polyethylene glycol and phosphates have been good phase formers, thus, binodal curve and tie lines construction were made possible. Since ABS is hindered by many factors. Therefore, the parameters such as volume ratio, molecular weight, temperature, physiological pH, salt effect, centrifugal force and shaking, were evaluated. The optimal conditions investigated for the salt was 0.5 M NaCl at ambient temperature. The salt effect of the ammonium chloride and sodium chloride on the separation had a significant different at p < .05. The physiological pH investigated were possible with only alkaline condition and 7.5 was the optimal pH evaluated. Also, the centrifugal force and shaking maximal conditions were 1500 and 50 rpm respectively. The separation of gamma aminobutyric acid from crude cell with the optimal conditions was archived with the Lactobacillus brevis. At an initial concentration of 482 mg/L, the percentage yield obtained was 99%. The different biomass loading of GABA producing bacteria show an increasing partition coefficient with increasing yield 108% when higher biomass was loaded. This elucidates the effect of biomass loading amount favours the partitioning of the GABA in the top phase, meaning, GABA can be separated and purified with this method. Also, the recovery of gamma aminobutyric acid at the final concentration of L. brevis (14000 mg/L), had an actual yield of over 88%. The assimilation from yeast, presents a percentage yield of 78% from the different GABA stock concentration 0f 40% (w/v). The extraction efficiency was 0.95 and the purity attained was 100%. ATPS has been proven to be a promising tool for the recovery of GABA. Keywords: Aqueous-two phase, Gamma-aminobutyric acid, Polyethylene glycol, Derivatization neurotransmitter, 2-pyrrolidone, glutamate decarboxylase