Production, partial purification and characterization of alfa-L-rhamnosidase from Penicillium ulaiense

VERÓNICA BEATRIZ RAJAL; ALICIA GRACIELA CID; GUILLERMO ELLENRIEDER; CARLOS MARIO CUEVAS

WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY

Año: 2009 vol. 25 p. 1025 - 1033

0959-3993

Penicillium ulaiense is a post-harvest pathogenic fungus that attacks citrus fruits. The objective of this work was to study this microorganism as an a-L-rhamnosidase producer and to characterize it from P. ulaiense. The enzyme under study is used for different applications in food and beverage industries. a-L-Rhamnosidase was produced in a stirred-batch reactor using rhamnose as the main carbon source. The kinetic parameters for the growth of the fungi and for the enzyme production were calculated from the experimental values. A method for partial purification, including (NH4)2SO4 precipitation, incubation at pH 12 and DEAE-Sepharose chromatography yielded an enzyme with very low b-glucosidase activity. The pH and temperature optima were 5.0 and 60°C, respectively. The Michaelis-Menten constants for the hydrolysis of p-nitrophenyl-a-L-rhamnoside were Vmax = 26±4 IU ml-1 and Km = 11±2 mM. The enzyme showed good thermostability up to 60ºC and good operational stability in white wine. Co+2 affected positively the activity; EDTA, Mn2+, Mg2+, dithiotreitol and Cu2+ reduced the activity by different amounts, and Hg2+ completely inhibited the enzyme. The enzyme showed more activity on p-nitrophenyl-a-L-rhamnoside than on naringin. According to these results, this enzyme has potential for use in the food and pharmacy industries since P. ulaiense does not produce mycotoxins.