Cloning, Expression, Purification, Characterization of Mannose-6-Phosphate Isomerase

• Main Authors: Hung-Wei Hsiao, 蕭鴻偉
• Other Authors: Tsuei-Yun Fang
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/59252405947590206607

碩士 === 國立臺灣海洋大學 === 食品科學系 === 99 === Mannose-6-phosphate isomerase (MPI; EC 5.3.1.8) catalyzes the reversible aldose-ketose isomerization between L-ribose and L-ribulose. In this study, the Thermoanaerobacterium saccharolyticum NTOU1 mpi gene was PCR-cloned into pET-21b and pET101/D-TOPO, and then expressed in Escherichia coli. In the IPTG induction experiments, both of the pET-21b and pET101/D-TOPO showed the poor NTOU1-MPI expression. The partial codon optimization still couldn’t improve the NTOU1-MPI expression. The Thermotoga maritima DSM 3109 mpi encoding MPI was PCR-cloned into pET-21b and then expressed in Escherichia coli. The Tm-MPI was obtained in the presence of 0.05 mM IPTG induction. Tm-MPI was purified sequentially using heat treatment, nucleic acid precipitation, and anion-exchange chromatography. The specific activity and purification fold of Tm-MPI were 1.21 U/mg and 100 fold. The purified Tm-MPI showed an apparent optimal pH of 7 and an optimal temperature at 90℃. The enzyme was stable at pH values ranging from 7 to 9, and the activity was fully retained after a 2 h incubation at 80-95℃. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of 0.5 mM Cu2+. The kcat value of Tm-MPI was determined for L-ribose was 21.5 s-1, respectively, and the KM value was 237 mM.The kcat/KM value was 0.09 mM-1 s-1. The MPI from Thermotoga maritima DSM 3109 is the most thermostable MPI to date, and an acceptable purity after heat treatment, suggesting that this enzyme has the potential to be used in rare sugar production.