Glycosidases from O. oeni: cloning and characterization

Apart from the alcoholic and malolactic (MLF) fermentations that are crucial in the production of wine, a myriad of other changes occur during the transformation of must into wine. While the reduction in acidity is the major sensory change associated with MLF, the importance of changes in aroma profile, palate structure and colour are increasingly being recognized but jet poorly understood. During vinification compounds that stimulate our visual, olfactory, gustatory and tactile senses are either released, synthesized, degraded or modified. Aroma accounts for only a fraction (ca. 1g/L) of the ingredients in a wine, but its significance is much greater than expected from its quantity. Wine bouquet stems mainly from two sources: the grape stock and soils, and from the fermentations. Therefore, the winemaker strives either to extract as many of the aroma compounds present in the grape (e.g. by managing the mash time and temperature) or to make those hidden available to the senses by digestion of glycosidically bound terpenes (e.g. by MLF). Since many of these processes involve the action of enzymes like glycosidases, it is important to have a better understanding of the impact of these enzymes on the sensory attributes of wine. In the past only in silico studies have been performed on the glycosidase enzymes from wine lactic acid bacteria (LAB). In this project several glycosidases from wine LAB will be cloned and characterised. We intend to establish the first wine LAB cell collection from Austrian grapes, must and wines. Following isolation and identification, a biochemical screening for the active strains will be performed. Then the glycosidases from the most promising strains will be cloned following three strategies: 1) using the putative sequences recently published to produce appropriate PCR primers, 2) by producing DNA libraries from the selected active strains and 3) by sequencing the purified native glycosidases to produce the appropriate oligonucleotides for cloning of the specific glycosidases. Detailed biochemical characterisation studies will be performed of the different cloned enzymes. This shall fill the gap in knowledge that actually exists on the molecular and physicochemical parameters governing glycosidase reactions under winemaking conditions. From the experiments planned we expect: (1) to gain a better understanding of the glycosidases from O. oeni, and other wine LAB that work under the harsh conditions of wine (pH 3.5, 14% ethanol, high sugar concentration, influence of gluconolacton) and (2) to collect information about the physicochemical parameters governing the glycosidase reactions in wine. This project involves two dissertation students and several MSc./BSc. thesis students for a period of 3 years.

Apart from the alcoholic and malolactic (MLF) fermentations that are crucial in the production of wine, a myriad of other changes occur during the transformation of must into wine. While the reduction in acidity is the major sensory change associated with MLF, the importance of changes in aroma profile, palate structure and colour are increasingly being recognized but jet poorly understood. During vinification compounds that stimulate our visual, olfactory, gustatory and tactile senses are either released, synthesized, degraded or modified. Aroma accounts for only a fraction (ca. 1g/L) of the ingredients in a wine, but its significance is much greater than expected from its quantity. Wine bouquet stems mainly from two sources: the grape stock and soils, and from the fermentations. Therefore, the winemaker strives either to extract as many of the aroma compounds present in the grape (e.g. by managing the mash time and temperature) or to make those hidden available to the senses by digestion of glycosidically bound terpenes (e.g. by MLF). Since many of these processes involve the action of enzymes like glycosidases, it is important to have a better understanding of the impact of these enzymes on the sensory attributes of wine. In the past only in silico studies have been performed on the glycosidase enzymes from wine lactic acid bacteria (LAB). In this project several glycosidases from wine LAB will be cloned and characterised. We intend to establish the first wine LAB cell collection from Austrian grapes, must and wines. Following isolation and identification, a biochemical screening for the active strains will be performed. Then the glycosidases from the most promising strains will be cloned following three strategies: 1) using the putative sequences recently published to produce appropriate PCR primers, 2) by producing DNA libraries from the selected active strains and 3) by sequencing the purified native glycosidases to produce the appropriate oligonucleotides for cloning of the specific glycosidases. Detailed biochemical characterisation studies will be performed of the different cloned enzymes. This shall fill the gap in knowledge that actually exists on the molecular and physicochemical parameters governing glycosidase reactions under winemaking conditions. From the experiments planned we expect: (1) to gain a better understanding of the glycosidases from O. oeni, and other wine LAB that work under the harsh conditions of wine (pH 3.5, 14% ethanol, high sugar concentration, influence of gluconolacton) and (2) to collect information about the physicochemical parameters governing the glycosidase reactions in wine. This project involves two dissertation students and several MSc./BSc. thesis students for a period of 3 years.