Altering the substrate specificity of recombinant l-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1 to favor d-allose production.

l-Rhamnose isomerase (l-RhI) catalyzes rare sugar isomerization between aldoses and ketoses. In an attempt to alter the substrate specificity of Thermoanaerobacterium saccharolyticus NTOU1 l-RhI (TsRhI), residue Ile102 was changed to other polar or charged amino acid residues by site-directed mutagenesis. The results of activity-screening using different substrates indicate that I102N, I102Q, and I102R TsRhIs can increase the preference against d-allose in comparison with the wild-type enzyme. The catalytic efficiencies of the purified I102N, I102Q, and I102R TsRhIs against d-allose are 148 %, 277 %, and 191 %, respectively, of that of wild-type enzyme, while those against l-rhamnose are 100 %, 167 % and 87 %, respectively. Mutant I102N, I102Q, and I102R TsRhIs were noted to have the altered substrate specificity, and I102Q TsRhI has the highest catalytic efficiency against d-allose presumably through the formation of an additional hydrogen bond with d-allose. The purified wild-type and mutant TsRhIs were further used to produce d-allose from 100 g/L d-fructose in the presence of d-allulose 3-epimerase, and the yields can reach as high as 22 % d-allulose and 12 % d-allose upon equilibrium. I102Q TsRhI takes only around half of the time to reach the same 12 % d-allose yield, suggesting that this mutant enzyme has a potential to be applied in d-allose production.

Characterization of a thermophilic L-rhamnose isomerase from Caldicellulosiruptor saccharolyticus ATCC 43494.

L-Rhamnose isomerase (EC 5.3.1.14, l-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-rhi gene encoding L-RhI was PCR-cloned from Caldicellulosiruptor saccharolyticus ATCC 43494 and then expressed in Escherichia coli. A high yield of active L-RhI, 3010 U/g of wet cells, was obtained after 20 °C induction for 20 h. The enzyme was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 90 °C. The enzyme was stable at pH values ranging from 4 to 11 and retained >90% activity after a 6 h incubation at 80 °C and pH 7-8. Compared with other previously characterized L-RhIs, the L-RhI from C. saccharolyticus ATCC 43494 has a good thermostability, the widest pH-stable range, and the highest catalytic efficiencies (k(cat)/K(M)) against L-rhamnose, L-lyxose, L-mannose, D-allose, and D-ribose, suggesting that this enzyme has the potential to be applied in rare sugar production.

Characterization of a thermophilic L-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1.

L-rhamnose isomerase (EC 5.3.1.14, L-RhI) catalyzes the reversible aldose-ketose isomerization between L-rhamnose and L-rhamnulose. In this study, the L-Rhi gene encoding L-Rhi was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. A high yield of the active L-RhI, 9780 U/g of wet cells, was obtained in the presence of 0.2 mM IPTG induction. L-RhI was purified sequentially using heat treatment, nucleic acid precipitation, and anion-exchange chromatography. The purified L-RhI showed an apparent optimal pH of 7 and an optimal temperature at 75 °C. The enzyme was stable at pH values ranging from 5 to 9, and the activity was fully retained after a 2 h incubation at 40-70 °C. L-RhI from T. saccharolyticum NTOU1 is the most thermostable L-RhI to date, and it has a high specific activity (163 U/mg) and an acceptable purity after heat treatment, suggesting that this enzyme has the potential to be used in rare sugar production.