The thermostable alpha-L-rhamnosidase RamA of Clostridium stercorarium: biochemical characterization and primary structure of a bacterial alpha-L-rhamnoside hydrolase, a new type of inverting glycoside hydrolase

An alpha-L-rhamnosidase clone was isolated from a genomic library of the th ermophilic anaerobic bacterium Clostridium stercorarium and its primary str ucture was determined. The recombinant gene product, RamA, was expressed in Escherichia coli, purified to homogeneity and characterized, It is a dimer of two identical subunits with a monomeric molecular mass of 95 kDa in SDS polyacrylamide gel electrophoresis, At pH 7.5 it is optimally active at 60 degrees C and insensitive to moderate concentrations of Triton X100, ethan ol and EDTA. It hydrolysed p-nitrophenyl-alpha-L-rhamnopyranoside, naringin and hesperidin with a specific activity of 82, 1.5 and 0.46 U mg(-1) respe ctively. Hydrolysis occurs by inversion of the anomeric configuration as de tected using H-1-NMR, indicating a single displacement mechanism. Naringin was hydrolysed to rhamnose and prunin, which could further be degraded by i ncubation with a thermostable beta-glucosidase. The secondary structure of RamA consists of 27% alpha-helices and 50% beta-sheets, as detected by circ ular dichroism, The primary structure of the ramA gene has no similarity to other glycoside hydrolase sequences and possibly is the first member of a new enzyme family.