XXXV Reunião Anual da SBBqResumoID:8037



 Purification and characterization of Saccharomycopsis fibuligera a-glucosidase with ability to form trehalose by transglycosylation


Giannesi, G.C.; Polizeli, M.L.T.M.; Terenzi, H.F.; Jorge, J.A



Depto. de Biologia, FFCLRP-USP., Ribeirão Preto, SP



 a-Glucosidase (a -D-glucoside glucohydrolase) form a group of exo-acting glycoside hydrolases with ample specificity, which release a -D-glucose from the non-reducing end of a -linked substrates. A number of a -glucosidases also catalyze the transglycosylation reaction producing a -glycosylated compounds. Saccharomycopsis fibuligera is known as a good trehalose producer. Preliminary studies showed the presence of a maltase activity that produced trehalose from maltose by transglycosylation. In the present report we describe the purification and some properties of a mycelial enzyme of S. fibuligera grown on starch as carbon source. The purification procedure consisted of precipitation with acetone, and DEAE-cellulose and Octyl-Sepharose chromatography. After the Octyl-Sepharose step, the specific activity was 14U/mg protein and 37-fold purification was achieved, with a yield of 65%. PAGE analysis showed a single band stained with Coomassie Blue. Analysis by SDS-PAGE showed two polypeptide bands, suggesting that the native enzyme was composed of two subunits with 105 kDa and 119 kDa. Maltase activity was maximal at pH 5.0 and 45oC. The Km and Vmax values for maltose of the purified enzyme were 0.46 mM and 18 U/mg protein, respectively. Although the enzyme preferentially hydrolyzed maltose, it also cleaved amylose, starch and maltooligosaccharides, but not p-nitrophenyl a -glucopyranoside, suggesting that the enzyme was a typical maltase. a -Glucosidase activity on maltose was inhibited by trehalose. At a higher concentration (5%) maltose was converted into trehalose and other maltooligosaccharides by transglycosylation reactions, as detected by TLC analysis. Maltooligosaccharides did not act as substrates for transglycosylation reaction. The mechanism of synthesis of trehalose and maltooligosaccharides by S. fibuligera is not yet completely elucidated. However, the present observations suggested that the enzyme produced trehalose directly from maltose. Finally, we would like to suggest that the transglycosylation activity exhibited by S. fibuligera a -glucosidase might explain the large level of trehalose produced by this organism grown on starch or maltose. Supported by CNPq, CAPES and FAPESP.