Application of Doehlert Experimental Design for Optimization of the Production of an Alkalophilic a -L-Rhamnosidase from Verticillium tenerum

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

Saltillo, Mexico

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

Sociedad Mexicana de Biotecnología y Bioingeniería. Presentación Oral

a-L-Rhamnosidases (Rhases, EC 3.2.1.40) catalyze the hydrolysis of terminal non-reducing a-L-rhamnose residues in both natural and synthetic a-L-rhamnosides (heteropolysaccharides, flavonoids and aromatic compounds) releasing free rhamnose (6-deoxy-L-manose). V. tenerum, an alkali tolerant filamentous fungus isolated from coastal region of Magdalena, Buenos Aires Province, Argentina, produces an extracellular Rhase active under alkaline conditions. The enzyme is expressed by V. tenerum growing in culture media containing V8® juice as carbon and energy source (CES) but the activity is low even with the addition of naringin as inducer. Replacement of V8 juice by soybean flour (SF), a powerful unspecific enzyme inducer, as CES produces an increment in the Rhase expression. In this study, the combined effects and optimum concentrations of SF and pancreatic casein hydrolysate (Tryptein, T) for Rhase production in batch cultures are investigated by using a response surface methodology. Experiments were carried out according to a Doehlert matrix centered in 1.5 g/L of SF and 3.75 g/L of T, covering the range between 1.25 and 6.25 g/L of SF, and 1.0 and 2.0 g/L of T. Cultures were carried out in Erlenmeyers-flasks (200 rpm, 30°C) for 15 days. The composition of the culture media was as follows (g/L): variable concentrations of SF and T; MgSO4 . 7H2O 0.5, KCl 0.5, FeSO4 . 7H2O 0.01, citric acid 0.01, K2HPO4 1.0, NaCO3 1.0, naringin 1.25 and antibiotic solution (streptomycin 25.0 g/l plus chloramphenicol 10.0 g/l) 4 ml. Enzymatic activity was determined at 37°C, for 30 min, using PNP-rhamnopyranoside as substrate. Substrate was prepared in TRIS-HCl 20 mM, pH: 9. A maximum in enzyme activity was obtained in the region of 3.75 g/L of T and 1 g/L of SF. An increase of 6 times was obtained from the initial conditions and 20 times from V8 medium. The highest enzyme activity was produced in 13 days of culture, and then it declined. The response surface methodology utilized proved to be a useful tool for the optimization of medium components for the production of Rhase.   (Key words: Verticillium tenerum, rhamnosidase, Doehlert experimental design)