Identification of the amino acid residues that change the substrate specificity of PHA synthase 1 from Pseudomonas putida GPo1

• Main Authors: Pei-Chien Tsai, 蔡佩倩
• Other Authors: Chia-Yin Lee
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/05311529025531561421

碩士 === 國立臺灣大學 === 農業化學研究所 === 92 === In our previously study, the substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (class II) from Pseudomonas putida GPo1 was successfully altered by localized semi-random mutagenesis. The evolved enzyme PS-E1, in which 14 amino acids had been changed exhibited broad substrate specificity. The promising amino acid positions which change the substrate specificity of PhaC1Pp were investigated by site directed mutagenesis. Mutations at L484V or S325C were remarkably enhanced the short-chain-length monomer composition up to 40 mol% in PHA accumulation experiment. Simultaneously mutated at S325C and Q481M of PhaC1Pp lead to scl monomer composition further enhanced up to 50 mol%. Furthermore, mutations at S482G, Q481M and A547V would increase PHA yields from 10 mol﹪(wild type ) to 20%, 35%, and 37%, respectively. Saturation mutagenesis experiment will be applied to further acquiring more information about the substrate specificity on PHA synthase 1.