Intracellular Localization And Characterization Of β-D-glucosidase From Lactobacillus Plantarum

Aroma is one of the important factors to the wine flavor and quality, and aromasubstances mainly present in free and bound glycoside forms in wine. In general, boundglycoside forms are more abundant than the free ones, which can be hydrolyzed withreleasing volatile aroma substances. β-D-glucosidase is a key enzyme in the enzymatic releaseof these bound monoterpenols from their glycosidic precursors, and much research hasfocused on β-D-glucosidases from LAB (mainly Oenococcus oeni) in malolactic fermentation(MLF). However, β-D-glucosidase activity was detected in Lactobacillus Plantarum, whichwas even higher than that in Oenococcus oeni. In addition, it was found that L. plantarumpossesses important oenological characteristics and could be as the next generation starterculture for MLF.In the present study, three strains of L. plantarum with high β-D-glycosidase activitywere obtained from five L. plantarum strains with p-nitro-phenyl-β-D-glucopyranoside(pNP-G) as substrate and their cellular localizations and assessment of activity ofβ-D-glucosidase were tested. Based on the previous study, the effect of wine parameters onstrains growth and β-D-glucosidase activity was investigated and the strain considered as apromising candidate for winemaking was gained by determining the enzymatic activity underthe combination of factors. Finally, its β-D-glucosidase properties were analyzed.(1) β-D-glucosidase was intracellular form for L. plantarum520, XJ-25, XJ-14, most ofwhich was insoluble protein existing in the cell membrane of spheroplast, and some wassoluble existing in periplasm and cytoplasm. Enzyme induction assay exhibitedβ-D-glucosidase of three strains was constitutive, however, the improvement of enzymaticactivities were observed when bacteria was cultured with arbutin or cellobiose. In addition,the improved MRS was verified as the most optimal medium.(2) β-D-glucosidase activity of L. plantarum520, XJ-25, XJ-14was inhibited under thewinemaking conditions except that these strains were incubated in the mediums with differentconcentrations of SO2. However, L. plantarum XJ-25grew well under the combinedconditions and its β-D-glucosidase displayed the highest enzymatic activity, which could beused as a promising candidate for the improvement of the wines aroma in winemaking. (3) With β-D-glucosidase having general heat resistance and stronger acid resistance, theoptimal temperature and pH of β-D-glucosidase of L. plantarum XJ-25were45℃and5.0,respectively. The effect of different metal ions on β-D-glucosidase varied, and the inhibitionof the enzyme was also showed under different concentrations of glucose, fructose or winerelated compounds. In addition, the enzyme had a high apparent affinity for pNP-G.