title: 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 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 Ca(2+), Mg(2+), Mn(2+), and Co(2+) nor reducing agents such as β-mercaptoethanol and dithiothreitol showed any effect on enzyme activity, whereas this activity was completely inhibited by Zn(2+) 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.
authors.affiliation: Centro de Investigación y Desarrollo en Fermentaciones Industriales, CINDEFI (UNLP; CONICET La Plata), Calle 50 y 115, B1900ASH, La Plata, Argentina. rojas@biotec.org.ar
authors.forename: Natalia Lorena
authors.initials: NL
authors.lastname: Rojas
authors.forename: Claudio Enrique
authors.initials: CE
authors.lastname: Voget
authors.forename: Roque Alberto
authors.initials: RA
authors.lastname: Hours
authors.forename: Sebastián Fernando
authors.initials: SF
authors.lastname: Cavalitto
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D055162:Biocatalysis; D004591:Electrophoresis, Polyacrylamide Gel; D044950:Flavanones / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006569:Hesperidin / Q000378:metabolism; D006868:Hydrolysis; D008970:Molecular Weight; D012210:Rhamnose / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D044950:Flavanones; D005960:Glucosides; D006569:Hesperidin; D006026:Glycoside Hydrolases; C022525:alpha-L-rhamnosidase; C005274:naringin; D012210:Rhamnose