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

NATALIA LORENA ROJAS, CLAUDIO ENRIQUE VOGET, ROQUE ALBERTO HOURS, SEBASTIÁN FERNANDO CAVALITTO

JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY

SPRINGER HEIDELBERG

Lugar: HEIDELBERG; Año: 2011 vol. 38 p. 1515 - 1522

1367-5435

Rhamnosidases are enzymes that catalyze the hydrolysis of terminal nonreducing 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 an efficient, simple, and inexpensive method for purifying the A. luteo albus rhamnosidase and describe the characteristics of the purified enzyme. In the presence of rhamnose as the sole carbon source, this fungus produces a rhamnosidase with a molecular weight of 109 kDa and a pI value of 4.6, as determined by SDS–PAGE and analytical isoelectric focusing, respectively. This enzyme was purified to homogeneity by chromatographic and electrophoretic techniques. Using p-nitrofenil-α-L-rhamnopiranoside as substrate, the enzyme activity showed pH and temperature optima of 8.0 and 55°C, respectively. The enzyme exhibited Michaelis–Menten kinetics, with K M and V max values of 3.38 mmol l−1 and 68.5 mmol l−1 min−1, respectively. Neither divalent cations such as Ca2+, Mg2+, Mn2+, and Co2+ nor reducing agents such as β-mercaptoethanol and dithiothreitol showed any effect on enzyme activity, whereas this activity was completely inhibited by Zn2+ at a concentration of 0.2 mM. This enzyme showed the capacity to hydrolyze some natural rhamnoglucosides such as hesperidin, naringin and quercitrin under alkaline conditions. Based on these results, and mainly due to the high activity of the A. luteo albus rhamnosidase under alkaline conditions, this enzyme should be considered a potential new biocatalyst for industrial applications.