Expression And Characterization Of A Thermostable Glucose Isomerase From The Hyperthermophile, Thermotoga Maritima

The glucose isomerase produced from Thermotoga maritima, a hyperthermophilic anaerobic bacterium, has a large potential in industrial application because of its excellent thermostability. T. maritima only can produce small amount glucose isomerase since the rigorous cultivation conditions. The glucose isomerase gene xylA from T. maritima was cloned and expressed in Escherichia coli using a heat-shock expression vector pHsh. We used the site directed mutagenesis of pHsh-xylA1 to change the energy of local mRNA secondary structures for higher expression. The result shows that 1 ml LB medium of E. coli JM109 harboring phsh-xylA2 can produce 4.95 U/mg GI, more than phsh-xylA1 which was only 1.3 U/mg.The recombinant glucose isomerase was purified 8.02-fold from the E. coli JM109 recombinant with a recovery of 49.02% using heat treatment and ion exchange chromatography, to give a single band on SDS-PAGE. The optimum pH and temperature of the enzyme were found to be pH 7.0 and 95℃. The enzyme was stable in the range pH 6～9, and showed a half-life of over 5 h at 95℃. The enzyme activity was activated with 1mmol/L Mg2+ and Co2+. The apparent Km and Vmax values for glucose were 105 mmol/L and 45.2 U/mg, respectively.The conditions for E. coli JM109(pHsh-xylA2) cell growth and expression in the LB medium in shake flask were optimized.The optimized medium is composed of:glucose 10 g/L, (NH4)2SO4 6 g/L,peptone 2 g/L, KH2PO4 10 g/L, MgSO4·7H2O 3 g/L, citric acid 3 g/L, trace elements solution(TES) 1 mL/L, pH 7.0. The effect of dissolved oxygen (DO) concentration and the feeding of carbon and nitrogen source for the growth and expression of E. coli JM109 (pHsh-xylA2) were studied at a 3.8L fermentor.Undering the controlling the DO above 30%, when carbon and nitrogen source was feeded at the middle log phase and temperature- heightening induction was performed at the late log phase, the ultimate optical density (OD600) of the cell culture was 44.8, the recombined GI activity was 4.23U/mg.