Purification and characterization of a novel alkaline α-L-rhamnosidase produced by Acrostalagmus luteo albus

Abstract

Rhamnosidases are enzymes that catalyze the hydrolysis of terminal non reducing L-rhamnose for the bioconversion of natural or synthetic rhamnosides. They are of great significance in the current biotechnological area with applications in food and pharmaceutical industrial processes. In this study we isolated and characterized a novel alkaline rhamnosidase from Acrostalagmus luteo albus, an alkali tolerant soil fungus from Argentina. We also present here an efficient, simple, and inexpensive method for purifying the A. luteo albus rhamnosidase and describe the characteristics of the purified enzyme. In presence of rhamnose as sole carbon source, this fungus produces a rhamnosidase of 109 kDa molecular weight and a pI value of 4.6 determined by SDS-PAGE and analytical isoelectric focusing, respectively. This enzyme was purified to homogeneity by chromatographic and electroforetic techniques. Using p-nitrofenil--L rhamnopiranoside as substrate, the enzyme activity shows pH and temperature optima of 8.0 and 55 ºC, respectively. The enzyme exhibited Michaelis-Menten kinetics with KM and Vmax values of 3.38 mmol.l-1 and 68.5 mmol.l-1.min-1. Divalent cations such as Ca+2, Mg+2, Mn+2, and Co+2 or reducing agents such as -mercaptoethanol and dithiothreitol showed no effect over enzyme activity, whereas this was completely inhibited by Zn+2 at a concentration of 0.2 mM. This enzyme showed the capability to hydrolyze some natural rhamnoglucosides such as hesperidin, naringin and quercitrin under alkaline conditions. On the basis of these results, and mainly due to the high activity of the A. luteo albus rhamnosidase under alkaline conditions, this enzyme should be considered as a potential new biocatalyst for industrial applications.