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Characterization of Two Distinct Glycosyl Hydrolase Family 78 α-L-Rhamnosidases from Pediococcus acidilactici

Author:
Michlmayr, Herbert, Brandes, Walter, Eder, Reinhard, Schümann, Christina, del Hierro, Andrés M., Kulbe, Klaus D.
Source:
Applied and environmental microbiology 2011 v.77 no.18 pp. 6524-6530
ISSN:
0099-2240
Subject:
Pediococcus acidilactici, acidity, alpha-L-rhamnosidase, citronellol, disaccharides, ethanol, fungi, genes, geraniol, glucose, hesperidin, hydrolysis, linalool, naringin, odor compounds, oxides, rhamnose, winemaking, wines
Abstract:
α-L-Rhamnosidases play an important role in the hydrolysis of glycosylated aroma compounds (especially terpenes) from wine. Although several authors have demonstrated the enological importance of fungal rhamnosidases, the information on bacterial enzymes in this context is still limited. In order to fill this important gap, two putative rhamnosidase genes (ram and ram2) from Pediococcus acidilactici DSM 20284 were heterologously expressed, and the respective gene products were characterized. In combination with a bacterial β-glucosidase, both enzymes released the monoterpenes linalool and cis-linalool oxide from a muscat wine extract under ideal conditions. Additionally, Ram could release significant amounts of geraniol and citronellol/nerol. Nevertheless, the potential enological value of these enzymes is limited by the strong negative effects of acidity and ethanol on the activities of Ram and Ram2. Therefore, a direct application in winemaking seems unlikely. Although both enzymes are members of the same glycosyl hydrolase family (GH 78), our results clearly suggest the distinct functionalities of Ram and Ram2, probably representing two subclasses within GH 78: Ram could efficiently hydrolyze only the synthetic substrate p-nitrophenyl-α-L-rhamnopyranoside (Vmax = 243 U mg–1). In contrast, Ram2 displayed considerable specificity toward hesperidin (Vmax = 34 U mg–1) and, especially, rutinose (Vmax = 1,200 U mg–1), a disaccharide composed of glucose and rhamnose. Both enzymes were unable to hydrolyze the flavanone glycoside naringin. Interestingly, both enzymes displayed indications of positive substrate cooperativity. This study presents detailed kinetic data on two novel rhamnosidases, which could be relevant for the further study of bacterial glycosidases.
Agid:
566840