| Flavor is one important factor to high quality wine. In addition to the initial impact thatwine color and taste have on the perception of wine quality, it is the aroma and flavor that hasthe greatest impact on consumers. β-D-glucosidase derived from Oenococcus oeni strains isthe most important glycosidase to hydrolyze glycosylated aroma precursors, releasing activearoma and flavor compounds, during malolactic fermentation (MLF). O. oeni SD-2a and31MBR are two excellent bacteria used in winemaking, possessing important oenologicalcharacteristics, particularly, being able to perform MLF effectively in winery conditions. Thus,investigating β-D-glucosidase of the two strains is of great significance for aromaenhancement and wine quality improvement in winemaking.In the present study, β-D-glucosidase activity of SD-2a and31MBR was detected,localized and characterized spectrophotometrically with synthetic substrate p-nitrophenylβ-D-glucopyranoside firstly. Then the ability of the two strains to hydrolyze glycosides wasalso evaluated with natural substrate extracted from grape in buffer and model wine. Finally,β-D-glucosidase genes of both strains were cloned, sequenced and characterized throughbioinformatic analysis, and the gene expression was analyzed with qRT-PCR technology. Themain results are as following.(1) Compared with MRST medium, ATB medium was preferable for O. oeni SD-2a and31MBR growth. Enzyme assay with synthetic substrate showed both SD-2a and31MBRpossessed β-D-glucosidase activity in ATB medium, with SD-2a showing higher activity than31MBR.(2) Enzyme localization assay showed31MBR possessed higher total enzyme activitythan that of SD-2a, while SD-2a showed higher whole cells activity; β-D-glucosidase wasintracellular form for both strains, most of which was soluble protein existing in cytoplasmand periplasm, and some was insoluble existing in protoplast membrane. Enzyme inductionassay exhibited β-D-glucosidase of the two strains was constitutive, however, varied enzymeactivities were observed when bacteria was cultured with arbutin or cellobiose.(3) The influence of winelike conditions and abiotic stress on β-D-glucosidase activity ofSD-2a and31MBR was conducted with synthetic substrate. Abiotic stress showed less effect on β-D-glucosidase activity of the two strains; crude β-D-glucosidase of both strains showedlittle activity under winelike conditions, while whole cells of SD-2a remained high activityunder such conditions.(4) The ability of SD-2a and31MBR to hydrolyze glycosides was also evaluated withnatural substrate extracted from grape. More glycosyl-glucose was detected for31MBR thanSD-2a in pH5.0buffer system; while in model wine system, more volatiles, whether onvariety or on content, was detected for SD-2a than31MBR, SD-2a proved possessing higherenzyme activity and preferable than31MBR for aroma enhancement during winemaking.(5) Five glucosidase genes were cloned from both SD-2a and31MBR, OEOE-1569coding for β-D-glucosidase and the others coding for6-P-β-glucosidase. Deduced amino acidsequences of sequenced genes OEOE-1210OEOE-1569and OEOE-0224showed highhomology (>99%) to the reported protein sequense in NCBI. Bioinformatic analysis showedthe glucosidases coded by these three genes were soluble protein, existing mainly incytoplasm.(6) The expression of five glucosidase genes cloned from both SD-2a and31MBR wasanalyzed with qRT-PCR technology. Gene OEOE-1569coding for β-D-glucosidase wasrelated to the intracellular β-D-glucosidase activity of the two strains, while gene OEOE-0224coding for6-P-β-glucosidase was related to whole cells β-D-glucosidase activity of bothstrains. The result confirmed the theory of carbon catabolite repression, and to some extentproved it is phosphoenolpyruvate dependent phospho transferase system (PEP-PTS) and6-P-β-glucosidase coded by gene OEOE-0224that contributes to whole cells β-D-glucosidaseactivity of SD-2a. |
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