Characterization and Immobilization of Recombinant L-Ribose Isomerase from Actinotalea fermentans ATCC 43279 and Altering Its Thermostability by Protein Engineering

• Main Authors: Wu, Tai-Jeng, 吳泰徵
• Other Authors: Fang, Tsuei-Yun
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/kpf779

碩士 === 國立臺灣海洋大學 === 食品科學系 === 105 === A putative L-ribose isomerase gene of A. fermentans ATCC 43279 was chemically synthesized, subcloned into pET-21b vector, and then overexpressed in Escherichia coli. The purified recombinant L-ribose isomerase (Af-RI) demonstrated its optimal activity at 45ºC and pH 8 (in Tricine-NaOH buffer). Metal ions are not required for L-ribose isomerase (L-RI) activity, but Hg2+ inhibits its activity completely. The enzyme has a half-life of 74 min at 50ºC and the equilibrium ratio of 30:70 between L-ribulose and L-ribose at 45ºC. The Vmax, kcat, KM, and catalytic efficiency of Af-RI against L-ribose are 232 U/mg, 6700 min-1, 31.3 mM, and 214 min-1·mM-1, respectively. The high expression yield of the Af-RI and its highest Vmax, kcat, and catalytic efficiency among the characterized recombinant L-RIs suggest that this recombinant enzyme shows a potential application to produce L-ribose in industry. To enhance the thermostability of Af-RI by mutagenesis, several mutations predicted by using the MAESTRO sever was constructed, and a recombinant Af-RI with a fused SUMO (small ubiquitin-related modifier) protein was also constructed. The crude extracts containing G39F, E61W, A68V, A68W and A165R mutant Af-RIs are no activity. The residual activities of the crude extracts containing wild-type, G176W, A233W, G43C/A238C Af-RIs and SUMO-Af-RI after incubating at 50ºC for 20 min are 19.0, 0, 16.2, 12.7 and 9.1%, respectively. It means that the thermostabilities of all mutant Af-RIs and SUMO-Af-RI aren’t better than that of wild-type Af-RI. Af-RI was overexpressed in strain E. coli ClearColi BL21 (DE3) which doesn’t produce endotoxin. The immobilized cells harboring Af-RI was prepared by entrapment with calcium alginate.The optimal conditions of immobilization are 0.05% Triton X-100, 5% alginate, 50 g/L cells, 200 mM CaCl2 solution, hardening in CaCl2 solution with 4 h. The immobilized cells harboring Af-RI showed its optimal activity at 50ºC and pH 8 (in Tricine-NaOH buffer) and have a half-life of 1386 min at 50ºC. The half-life of immobilized cells at 50ºC is about nineteen times longer than that of free enzyme.