| Summary: | 碩士 === 國立屏東科技大學 === 生物科技系所 === 101 === Gamma-aminobutyric acid (GABA) is a ubiquitous non-protein amino acid which is produced primarily by the -decarboxylation of glutamate catalyzed by the enzyme glutamate decarboxylase (GAD) in organisms. The functions of GABA include the inhibition of neural transduction, the regulation of cardiovascular functions such as blood pressure and heart rate, and the involvement in the sensations of pain and anxiety in vertebrate. It is well known that GABA functions in animals as a major inhibitory neurotransmitter in the central nervous system. Monascus purpureus is one of the most important fungus used for food processing and health foods development. Monascus species can produce several hydrolytic enzymes and secondary metabolites of high economic value, which are applied majorly in medicinal and biotech industries. For Monascus species possess special physiological activities and are generally regarded as safe, it is suggested to utilize its glutamate decarboxylase for the production of GABA. The purpose of this study is to clone the M. purpureus GAD gene for functional protein expression and enzyme activity analysis. Analysis of culture medium of M. purpureus from different incubation time shows that enzyme activity is positively related to incubation time. In other words, the enzyme activity of glutamate decarboxylase increases with days of culture. Degenerate primers are designed according to known sequences of glutamate decarboxylase from fungi for PCR cloning the GAD gene of M. purpureus. Sequence analysis indicates that it is 86% in similarity to the GAD gene of Aspergillus fumigatus Af293. It contains a pyridoxal 5'-phosphate hydrate (PLP) binding site, The GAD-A.ory and GAD-A.fum both belong to the DOPA decarboxylase family (DDC). Sixteen high copy number transformants are screened for GAD expression in Pichia pastoris. It can be applied for mass production of GABA as health foods in the future.
Keywords: glutamate decarboxylase, glutamate, -aminobutyric acid (GABA), Monascus purpureus
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