Purification and functional characterisation of an α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565

Abstract

An extracellular a-l-rhamnosidase from Penicillium citrinum MTCC-3565 has purified to homogeneity from its culture filtrate using ethanol precipitation and cation-exchange chromatography on carboxymethyl cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 45.0 kDa in SDS-PAGE analysis showing the purity of the enzyme preparation. The native PAGE analysis showed the monomeric nature of the purified enzyme. Using p-nitrophenyl a-l-rhamnopyranoside as substrate, K m and V max values of the enzyme were 0.30 mm and 27.0 lm min mg )1 , respectively. The k cat value was 20.1 s giving k cat ⁄ K m value of 67.0 mm s )1 for the same substrate. The pH and temperature optima of the enzyme were 8.5 and 50°C, respectively. The activation energy for the thermal denaturation of the enzyme was 29.9 KJ mol )1 . The a-l-rhamnosidase was able to hydrolyse naringin, rutin and hesperidin and liberated l-rhamnose, indicating that the purified enzyme can be used for the preparation of a-l-rhamnose and pharmaceutically important compounds by derhamnosylation of natural glycosides containing terminal a-lrhamnose. The a-l-rhamnosidase was active at the level of ethanol concentration present in wine, indicating that it can be used for improving wine aroma.