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

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

WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY

SPRINGER

Lugar: London; 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 alpha-l-rhamnosidase producer and to characterize it from P. ulaiense. The enzyme under study is used for different applications in food and beverage industries. alpha-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 beta-glucosidase activity. The pH and temperature optima were 5.0 and 60°C, respectively. The Michaelis Menten constants for the hydrolysis of p-nitrophenyl-alpha-l-rhamnoside were V max = 26 ± 4 IU ml-1 and K m = 11 ± 2 mM. The enzyme showed good thermostability up to 60°C and good operational stability in white wine. Co2+ 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-alpha-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.