Protein Engineering and Immobilization of Recombinant L-Ribose Isomerase from Actinotalea fermentans ATCC 43279 to Alter Its Thermostability

• Main Authors: Liou, Yu-Jiun, 劉俞均
• Other Authors: Fang, Tsuei-Yun
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/7qvf54

碩士 === 國立臺灣海洋大學 === 食品科學系 === 106 === L-Ribose isomerase (L-RI) catalyzes the reversible aldose-ketose isomerization between L-ribulose and L-ribose. L-ribose is an aldose which is not often found in nature. It can be used as a precursor material for antiviral and anticancer drugs. The gene of Actinotalea fermentans ATCC 43279 was overexpressed in Escherichia coli BL21 Star (DE3), the purified recombinant enzyme demonstrated its optimal activity at 45℃ and the half-life at 50℃ was 58 min. To enhance the thermostability of Af-RI by protein engineering, four residues (L69, P78, V139, F141) on the interface of enzyme tetramers and the mutation sites from the study of Geodermatophilus. obscurus DSM43160 have been chosen for mutagenesis (D135, A221). L69Q, P78K, P78R, P78T, V139T, F141Y, D135G, A221W, R132Q/D134G/D135G, P220Q/A221W and Af-RI-ATS were successfully constructed and expresses in E. coli BL21 Star (DE3). The relative activities of the crude extracts containing wild-type, F141Y, R132Q/D134G/D135G and P220Q/A221W was 100%, 77.6%, 58.4%, and 26.9%, respectively. After incubating at 50℃ for 10 min, the residual activities of wild-type and F141Y were 27% and 38.4%, respectively. After purification, the specific activity of F141Y Af-RI was 25.1 U/mg; the half-life at 50℃ was 66.6 min, which was 1.14 times of wild-type. The cells containing Af-RI were immobilized by entrapment in the beads of alginate and cured by treatment with CaCl2 solution, the immobilization efficiency was up to 67%. The optimum conditions are 1.2% alginate, 25 g/L cells, and 0.05 M CaCl2 solution. The immobilized cells showed its optimal activity at 45℃ and pH 8.0 (Tricine-NaOH buffer). The half-life of immobilized cells at 50℃ was 52.5 min, and the activity was about 50% after 16 days of storage at 4℃.