Two new thermostable α-l-rhamnosidases from a novel thermophilic bacterium

Two new thermostable α-l-rhamnosidases with novel substrate hydrolysis pattern were cloned and expressed from a new thermophilic bacterium. Fragments of the two α-l-rhamnosidase genes, rhmA and rhmB were identified in a partially sequenced genome of the bacterium. Whole genes were recovered by amplifying flanking sequences with single specific primers and nonspecific walking primers. The recovered genes were then cloned into Escherichia coli and their enzymes produced and purified. Both enzymes were dimers and the MW of the monomers, were 104 and 107 kDa for RhmA and RhmB, respectively. Both rhamnosidases had a temperature optimum at 70 °C. RhmA had pH optimum at 7.9 and RhmB had a broad pH optimum of 5.0 to 6.9 and RhmA had over 50% activity in the pH interval 5.0 to 8.7 and RhmB in the pH interval 4.0 to 7.9. Both enzymes had over 20% residual activity after 24-h incubation at 60 °C. RhmA and RhmB had Km values of 0.46 and 0.66 mM and Vmax values of 134 and 352 U mg−1 respectively, on p-nitrophenyl-α-l-rhamnopyranoside. Both rhamnosidases were active on both α-1,2- and α-1,6-linkages to beta-d-glucoside.