| Summary: | 碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 97 === Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is the first enzyme of the phenylpropanoid biosynthetic pathway. The enzyme catalyzes the non-oxidative deamination of phenylalanine to trans-cinnamic acid. This reaction leads to bio- synthesis of many phenylpropanoid-derived secondary metabolic products in plants, such as flavonoids and lignin.
In this study, PAL was purified from water bamboo by buffer extraction, ammonium sulfate precipitation, Sephacryl S-300, Phenyl- Sepharose, and Fast Protein Liquid Chromatography (FPLC). The purified PAL with 110.5-fold purification and specific activity of 53.3 nkat/mg protein obtained from water bamboo. Using Superose 6 and Superose 12 column (FPLC), the molecular weight of native form PAL was estimated to be 290-300 kD, the molecular weight of subunit form was determined to be 75 kD by SDS-PAGE.Using Western blot by antibody of bamboo PAL, and the results indicated that after the purification procedure the enzyme PAL had been purified. The pure enzyme was used to test biochemical properties.
PAL from water bamboo is similar with Gramineae plants in biochemical properties. The Km values for L-Phe were 516.The optimum temperature and pH were 45℃ and 8.5, respectively. The activation energy was 16.1 kcal/mol. Ca2+, Mg2+, Mn2+ actived PAL activity in low concentration; Co2+, Hg2+ inhibited PAL activity. Secondary metabolites including trans-cinnamic acid, p-coumaric acid, ferulic acid, p-amino-benzonic acid, and caffeic acid with phenomena of feedback inhibition. PAL has high substrate specificity to L-phenylalanine, and the presence of seryl, tyrosyl and histidyl groups was found to be essential for enzyme catalysis.
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