Biochemical characterisation of four rhamnosidases from thermophilic bacteria of the genera Thermotoga, Caldicellulosiruptor and Thermoclostridium.

Carbohydrate active enzymes are classified in databases based on sequence and structural similarity. However, their function can vary considerably within a similarity-based enzyme family, which makes biochemical characterisation indispensable to unravel their physiological role and to arrive at a meaningful annotation of the corresponding genes. In this study, we biochemically characterised the four related enzymes Tm_Ram106B, Tn_Ram106B, Cb_Ram106B and Ts_Ram106B from the thermophilic bacteria Thermotoga maritima MSB8, Thermotoga neapolitana Z2706-MC24, Caldicellulosiruptor bescii DSM 6725 and Thermoclostridium stercorarium DSM 8532, respectively, as α-L-rhamnosidases. Cobalt, nickel, manganese and magnesium ions stimulated while EDTA and EGTA inhibited all four enzymes. The kinetic parameters such as Km, Vmax and kcat were about average compared to other rhamnosidases. The enzymes were inhibited by rhamnose, with half-maximal inhibitory concentrations (IC50) between 5 mM and 8 mM. The α-L-rhamnosidases removed the terminal rhamnose moiety from the rutinoside in naringin, a natural flavonone glycoside. The Thermotoga sp. enzymes displayed the highest optimum temperatures and thermostabilities of all rhamnosidases reported to date. The four thermophilic and divalent ion-dependent rhamnosidases are the first biochemically characterised orthologous enzymes recently assigned to glycoside hydrolase family 106.