Aims: The objective of this study was to investigate what types of enzymes are being produced by non-Saccharomyces yeasts isolated from grapes in South Africa vineyards and clari®ed grape juice. These enzyme pro®les could pave the way for attributing speci®c effects in wine to some of these enzymes produced by so-called wild yeasts associated with grape must. Methods and Results: In this study 245 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces were screened for the production of extracellular pectinases, proteases b-glucanases, lichenases, b-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts, representing 21 species, were previously isolated from grapes and clari®ed grape juice. The production of all extracellular hydrolytic enzymes screened for was observed except b-glucosidase activity. The amount and range of enzymes produced varied with different isolates of the same species. Conclusion: This study clearly revealed the potential of non-Saccharomyces wine yeasts to produce a wide range of useful extracellular enzymes during the initial phase of wine fermentation. Signi®cance and Impact of the Study: Enzymes produced by indigenous yeasts associated with grapes and juice might be harnessed to catalyse desired biotransformations during wine fermentation.

We describe an improved, rapid and cost effective assay for measuring O6-alkylguanine-DNA alkyltransferase (AGT) levels in large numbers of small biological samples. The assay is based on the use of a synthetic O6-methylguanine oligonucleotide bound to magnetic beads via a biotin-streptavidin linkage and bound to its complement. A 35S label is incorporated into the free end of the duplex. The basis of the assay lies in the observation that the restriction enzyme PvuII will not cleave the bead-bound duplex oligonucleotide having an O6-methylguanine within the restriction sequence. However, on removal of the methyl group by AGT present in cell extracts prior to incubation with PvuII, the 35S-labelled oligonucleotide is cleaved by the restriction enzyme, releasing a 35S-labelled 8 bp fragment. Due to the stoichiometric nature of the AGT reaction, quantitation of AGT is easily achieved by counting an aliquot of the supernatant after pelleting the unrestricted bead complexes with a magnet. AGT levels measured in certain cell lines and human lymphocytes by the reported assay are comparable to other methods. The assay can be performed in basic laboratories and allows for the rapid processing of many samples simultaneously, which could prove useful in clinical and epidemiological studies.

Aims: The main objective of this study was to develop polysaccharide-degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking. Methods and Results: Two yeast expression ⁄ secretion gene cassettes were constructed, namely (i) a pectinase gene cassette (pPPK) consisting of the endo-polygalacturonase gene (pelE) from Erwinia chrysanthemi and the pectate lyase gene (peh1) from Erwinia carotovora and (ii) a glucanase ⁄ xylanase gene cassette (pEXS) containing the endo-b-1,4-glucanase gene (end1) from Butyrivibrio fibrisolvens and the endo-b-1,4-xylanase gene (xynC) from Aspergillus niger. The commercial wine yeast strain, VIN13, was transformed separately with these two gene cassettes and checked for the production of pectinase, glucanase and xylanase activities. Pinot Noir, Cinsaut and Muscat d'Alexandria grape juices were fermented using the VIN13[pPPK] pectinase-and the VIN13[pEXS] glucanase ⁄ xylanase-producing transformants. Chemical analyses of the resultant wines indicated that (i) the pectinase-producing strain caused a decrease in the concentration of phenolic compounds in Pinot Noir whereas the glucanase ⁄ xylanase-producing strain caused an increase in phenolic compounds presumably because of the degradation of the grape skins; (ii) the glucanase ⁄ xylanase-producing strain caused a decrease in wine turbidity, especially in Pinot Noir wine, as well as a clear increase in colour intensity and (iii) in the Muscat d'Alexandria and Cinsaut wines, the differences between the control wines (fermented with the untransformed VIN3 strain) and the wines produced by the two transformed strains were less prominent showing that the effect of these polysaccharide-degrading enzymes is cultivar-dependent. Conclusions: The recombinant wine yeasts producing pectinase, glucanase and xylanase activities during the fermentation of Pinot Noir, Cinsaut and Muscat d'Alexandria grape juice altered the chemical composition of the resultant wines in a way that such yeasts could potentially be used to improve the clarity, colour intensity and stability and aroma of wine. Significance and Impact of the Study: Aspects of commercial-scale wine processing and clarification, colour extraction and stabilization, and aroma enhancement could potentially be improved by the use of polysaccharidedegrading wine yeasts without the addition of expensive commercial enzyme preparations. This offers the potential to further improve the price : quality ratio of wine according to consumer expectations.