Purification And Characterization Of β-D-glucosidase From Oenococcus Oeni31MBR

Winemaking usually involves two biotransformation steps: an initial alcoholicfermentation step in which yeasts convert must into alcohol and an optional secondarymalolactic fermentation (MLF) step undertaken by lactic acid bacteria (LAB) to convert malicacid into lactic acid. MLF is an important stage in winemaking that is generally carried out byOenococcus oeni which induces many other transformations to modify the flavor of wine.Oenococcus oeni can not only survive harsh wine conditions, but also is a source of plentyenzymes.β-glucosidase hydrolyze odourless and nonvolatile glycosylated precursors duringaging, releasing aroma and volatile compounds to improve the aroma profile of wines.O. oeni31MBR, possessed high β-glycosidase activiy in the intact cell and breakingsupernatant, is usually the preferred species for MLF and one commercial strain prevalent inChina with an excellent performance to conduct MLF. The aim of this work was to purify andcharacterize β-glucosidase of O. oeni31MBR from its native crude extracts to understand theoenological properties of this enzyme. The main results are as following.（1）Response surface methodology was used to optimize ultrasonic conditions on the basisof single factor test. The results showed that optimum conditions were: ultrasonic time20min,ultrasonic power130W, and the cell concentration about OD600=2.0. Under such conditions,total β-D-glucosidase activity from31MBR was about13.89U, which was higher thanrepeated freezing and melting and glass-bead. β-Glucosidase can be extracted fromOenococcus oeni effectively by ultrasonic, and it is feasible to optimize ultrasonic extractionconditions using response surface methodology.（2）This study was designed to characterize a β-glucosidase from Oenococcus oeni31MBR. An intracellular β-glucosidase was partially purified using a combination ofammonium sulfate precipitation and chromatographic methods. A single band was obtained inSDS-PAGE electrophoresis, indicating that the enzyme was highly purified and had anestimated molecular mass of38.9kDa. The enzyme was partially purified up to7.7-fold to aspecific activity of0.115U/mg with yield of5.8%after4methods were applied. （3）The enzyme exhibited highest activity at pH4.5to5.0. The optimum temperature was45℃.4%~16%ethanol promoted the activity of this enzyme. The enzyme was partiallyinhibited by glucose, arabinose and xylose, while galactose and sucrose promoted enzymeactivity to some extent. Among the several metal ions assayed, Cr2+, Li+, Mn2+exhibited apartial promotion effect. Hg2+, Cu2+and Al3+inhibitated enzyme activity. The Km and Vmaxvalues for p-nitrophenyl-β-D-glucopyranoside(pNPG) were1.05mmol/L and0.957nmol/min,respectively.