Physico-kinetic and functional features of a novel β-glucosidase isolated from milk thistle (Silybum marianum Gaertn.) flower petals

A highly abundant beta-glucosidase from petals of Silybum marianum has been purified and characterized for its physico-kinetic properties. The 135 kDa enzyme was a homodimer with subunit molecular mass of 67.6 kDa. The characteristic catalytic properties of the enzyme included acidic pH optimum (5.5), meso-thermostability, and beta-linked substrate specificity with preference for gluco-conjugate but a marked (>50 %) activity with D-fuco-conjugates and considerable (similar to 16 %) activity towards D-galacto-conjugates. The enzyme showed high affinity for p-nitrophenyl glucoside (pNPG) with K-m and V-max values of 0.25 mM and 5.35 mu kat.mg(-1) enzyme protein. Thus, the enzyme had a very high (292,000 M-1.s(-1)) catalytic efficiency (K-cat/K-m). Thermal catalytic optimum of enzyme was 40 degrees C with activation energy value 8.26 kCal.Mol(-1). The enzyme showed significant insensitivity to D-gluconic acid lactone inhibition (57 % at 5 mM) with an apparent K-i 3.8 mM. The transglucosylating ability of enzyme was noticed for glucosylation of geraniol and withaferin-A with pNPG as glucosyl donor but cellobiose did not serve as the glycosyl donor. Partial proteomics of the enzyme revealed two peptide fragment sequences, VTPSNEVH and KRSEESNF. These motifs showed significant matching/sequence conservation with some other glycohydrolases. The novelties of purified enzyme hold potential to expand a library of catalytically characteristic members of the hydrolase family from plants for use in biotransformation applications.