Partial Purification and Characterization of an Alkalophilic a -L-Rhamnosidase from Verticillium tenerum

ROJAS, NATALIA LORENA; BLANCO FERNANDEZ, DOLORES; CAVALITTO, SEBASTIÁN FERNANDO; VOGET, CLAUDIO ENRIQUE; HOURS, ROQUE ALBERTO

Saltillo, Mexico

Congreso; Segundo Congreso Internacional Food Science and Food Biotechnology in Developing Countries; 2006

Sociedad Mexicana de Biotecnología y Bioingeniería

a-L-Rhamnosidase (a-Rha, EC 3.2.1.40) is an enzyme of considerable importance used for the bioconversion of natural or synthetic rhamnosides. The aim of this research is to study the purification of an alkaliphilic a-Rha produced by V. tenerum when grown in a medium containing soybean flour and naringin. Under these growing conditions V. tenerum also produces an b-glucosidase (b-Glu) which interferes in some applications of this enzyme. A crude extract of V. tenerum was desalted on a Sephadex™ G-25 column using Tris-HCl (20 mM, pH: 7.0) buffer. Desalted proteins were applied onto a Q Sepharose™ column. A NaCl gradient (0-1 M) in the same buffer was used. Bound fractions were screened toward p-nitrophenyl-a-L-rhamnopyranoside; a synthetic substrate for a-Rha activity. Active fractions were pooled and applied onto a Sephacryl™ S-100 HR column, pH 9.0. As result, a-Rha was partially purified with an overall activity recovery of 30%. Although a-Rha specific activity increased during this purification process, no satisfactory separation of a-Rha from b-Glu could be obtained even using ion exchange chromatography at different pH values (using salt gradients). Gel filtration and hydrophobic chromatographies were also not effective. In order to determine the enzyme pH and temperature stability, a Doehlert design was used to represent the combined effects by means of a polynomial model. Then, a response surface methodology was applied to represent these effects. Experiments were carried out according to a Doehlert matrix, at pH: 6-11 and 37-60°C. Nevertheless, under the conditions mentioned above for the stability assay, b-Glu was inactivated after incubating the final extract (3 h, at 48°C), while more than 70% of a-Rha activity remained. It was also found that above 50°C both enzyme activities were completely lost, and that the enzyme activities also declined when pH decreased. Therefore, the inactivated fraction containing residual a-Rha could be used for further characterization experiments and to evaluate its activity towards naringin and other natural substrates without b-Glu interferences. The separation of the two enzymes with apparently similar pI and MW is currently under investigation.