Altering the substrate specificity of recombinant L-rhamnose isomerase from Thermoanaerobacterium saccharolyticum NTOU1 to favor D-allose production

Abstract

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.