Enzymes in Winemaking: Harnessing Natural Catalysts for Efficient Biotransformations - A Review P. van Rensburg and I.S. Pretorius Institute for Wine Biotechnology and Department of Viticulture & Oenology, University of Stellenbosch, Private Bag X1, 7602 Matieland (Stellenbosch), South Africa Submitted for publication: July 2000 Accepted for publication: August 2000 Key words: Enzymes, glucanases, glycosidases, glucose oxidases, pectinases, proteases, ureases, wine yeas Enzymes play a definitive role in the ancient and complex process of winemaking. From a scientific and technical point of view, wine can be seen as the product of enzymatic transformation of grape juice. From the pre-fermenta- tion stage, through fermentation, post-fermentation and aging, enzymes are the major driving forces catalysing var- ious biotransformation reactions. These biocatalysts originate not only from the grape itself but also from yeasts and other microbes (fungi and bacteria) associated with vineyards and wine cellars. Through better understanding of these enzymatic activities, winemakers have come to learn how to control the unwanted enzymes while optimis- ing the desired activities. Today, winemakers reinforce and extend the action of these endogenous enzymes by the judicious application of an ever-increasing spectrum of commercial enzyme preparations. These enzyme prepara- tions are applied to winemaking with the aims of improving the clarification and processing of wine, releasing vari- etal aromas from precursor compounds, reducing ethyl carbamate formation and lowering alcohol levels. This review article summarises the most important enzymes applied to winemaking, the nature and structure of their substrates, and the reactions catalysed by these enzymes. This paper also reviews the limitations of the endogenous enzymes derived from grapes and microbes present in must and wine, along with the effects of commercial enzyme preparations on process technology and the quality of the final product. Prospects of developing wine yeast strains expressing tailored enzymes are also highlighted. INTRODUCTION The term "enzyme" is derived from the Latin words meaning "in yeast". Enzymes were once thought to exist at organism level, until in 1926 Sumner demonstrated that enzymes are in fact pro- teins which act as biological catalysts. Apart from facilitating reactions they are also able to accelerate reactions without under- going any permanent structural change (Underkofler, 1976). While the types of reactions catalysed by enzymes are limited (hydrolyses, oxidations, reductions, etc.) their numbers remain very high. This is due to one of the most intriguing characteristics of enzymes, their specificity. Enzymes have the capacity to act on one substance, or a limited number of substances, by recognising only a specific chemical group or substrate. This absolute speci- ficity may be broadened or removed completely by a small mod- ification of the enzyme which is sufficient to render it inactive. While these characteristics may limit the field of application, they also provide for targeted interventions, which could not normally be achieved in any other way. Enzymes play a pivotal role in the winemaking process. In addition to enzymes which occur in pre- and post-fermentation practices, there are at least ten different enzymes driving the fer- mentation kinetics that convert grape juice to wine. It is therefore of key importance to understand the nature and behaviour of these enzymes and to create the optimal conditions to exploit those enzymes which are beneficial, while inhibiting those which may be detrimental to wine quality. Many of these enzymes orig- inate from the grape itself, the indigenous microflora on the grape and the microorganisms present during winemaking. Since the endogenous enzymes of grapes, yeasts and other microorganisms present in must and wine are often neither efficient nor sufficient under winemaking conditions to effectively catalyse the various biotransformation reactions, commercial enzyme preparations are widely used as supplements. All these commercial enzyme preparations are obtained from microorganisms cultivated on substrates under conditions that optimise their production and facilitate their purification at a competitive cost. Research in this field is very active and contin- ually expanding. The number of enzymes produced on an indus- trial scale (approximately 30) represents only a fraction of the total number of enzymes that have been discovered: there are 2500 different enzyme-catalysed reactions listed in the International Union Handbook of Enzymes Nomenclature (Gacesa & Hubble, 1998). This article summarises the most important enzymes that act in winemaking to improve (i) the clarification and processing of wine (pectinases, glucanases, xylanases, proteases), (ii) the release of varietal aromas from precursor compounds (glycosi- dases), (iii) the reduction of ethyl carbamate formation (urease), and (iv) the reduction in alcohol levels (glucose oxidase). For a better understanding of the reactions catalysed by these enzymes, some background information is presented on the nature and structure of various substrates transformed by the enzymes. This paper also reviews the limitations of the endogenous enzymes derived from grapes or microbes in must and wine (Table 1), along with the advantages resulting from application of industri- al enzyme preparations. Prospects for developing wine yeast strains expressing these heterologous enzymes are also highlighted. Acknowledgements: We are grateful to the South African Wine Indusu:y (Winetech) and the National Research Foundation (NRF) forfinancial support. The authors thank T. Plantingafor criti- cal reading of this manuscript. S. Afr. J. Enol. Vitic., Vol. 21, Special Issue, 2000 52