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Cloning and expression of phenylalanine dehydrogenase gene from Bacillus lentus |
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| รหัสดีโอไอ | |
| Title | Cloning and expression of phenylalanine dehydrogenase gene from Bacillus lentus |
| Creator | Mayura Thongchuang |
| Contributor | Kanoktip Packdibamrung |
| Publisher | Chulalongkorn University |
| Publication Year | 2549 |
| Keyword | Genes, Cloning, Gene expression, Amino acid sequence, ยีน, โคลนิง, ลำดับกรดอะมิโน |
| Abstract | NAD⁺-dependent phenylalanine dehydrogenase (EC 1.4.1.20) catalyzes the reversible oxidative deamination of L-phenylalanine to form ammonia, phenylpyruvate and NADH. Our research group has studied phenylalanine dehydrogenase from Bacillus lentus and found that the enzyme had high substrate specificity in the oxidative deamination on L-phenylalanine and the reductive amination on phenylpyruvate. No loss of the enzyme activity was observed upon incubation at 50° for 4 hours. The enzyme retained 50% of the activity after incubation at the same temperature for 3 days. Therefore, in this research, the amino acid sequence of 3 internal peptide fragments of phenylalanine dehydrogenase were determined and used for degenerated primer design. The nucleotide sequencing of phenylalanine dehydrogenase gene (phedh) was investigated by inverse PCR and then the whole gene was cloned and expressed in E. coli BL2 (DE3) using expression vector, pET-17b. The specific activity of crude recombinant enzyme was 92.0 fold higher than that of the enzyme from B. lentus. The optimum condition for phedh gene expression was induction with 0.2 mM IPTG for 8 hours. In spite of daily subculture for 50 days, the phedh gene expression in E. coli BL21(DE3) remained 57.7% of that of the parent. Recombinant enzyme was purified 2.11 fold with a 31.0% yield by procedure involving 40-50% ammonium sulfate precipitation, DEAE-Toyopearl and Butyl-Toyopearl column chromatography. The enzyme had a molecular mass about 340 kDa and consisted of 8 identical subunits. The optimum pHs for the oxidative deamination and the reductive amination were 10.7 and 7.8 respectively and optimum temperatures were 45[degrees Celsius] and 50[degrees Celsius], respectively. The enzyme was stable over a broad pH range from 7.0 to 11.0 The apparent K[subscriptm] values for L-phenylalanine, NAD⁺, phenylpyruvate, NH₄CI and NADH were 0.45, 0.40, 0.15, 48 and 0.15 mM, respectively. |
| URL Website | cuir.car.chula.ac.th |
