The compound ethyl carbamate or urethane is a versatile carcinogen that can induce cancer in various organisms (Sotomayor and Collins, 1990).

It occurs naturally in fermented beverages such as beer, sake and wine and in fermented foods such as olives and yoghurt (Ough, 1976). It was also detected in cheese, tea, soy sauce and bread, with the levels of ethyl carbamate in toasted bread being six times higher than in the fresh counterpart. Traditionally, ethyl carbamate has been used as an intermediate in the synthetic preparation and modification of resins and as a solvent for pesticides, fumigants and cosmetics. It is also known for its narcotic action and was used for many years as an anaesthetic (Mirvish, 1968).

In 1985 the Liquor Control Board of Ontario, Canada, discovered large quantities (up to 13 400 mg/l) of ethyl carbamate in sherries, dessert wines and distilled spirits. This triggered worldwide concern for consumer health and lead to the introduction of regulatory limits of ethyl carbamate in various types of alcoholic beverages.

Precursors of ethyl carbamate. The most important precursor in wine is believed to be the urea produced during the metabolism of arginine by the wine yeast Saccharomyces cerevisiae (Monteiro et al, 1989). l-Arginine is one of the predominant amino acids present in grape juice and is degraded by the wine yeast to ornithine, ammonia and carbon dioxide during fermentation, by means of the arginase enzyme (Figure 1) . Urea is produced as an intermediate product and since it is a denaturing agent, the yeast secretes the excess urea into the grape must. This takes place via a facilitated diffusion system as soon as the urea reaches a specific concentration inside the cell. Ethanol produced during the wine fermentation reacts with the urea in the fermenting grape must and ethyl carbamate is formed. Other less important precursors of ethyl carbamate are citrulline and carbamyl phosphate (Ough et al, 1988).

Other factors determining ethyl carbamate production. It is known that wine can show a considerable increase in ethyl carbamate content during storage (Stevens and Ough, 1993). Furthermore, an increase in storage temperature results in a dramatic increase in ethyl carbamate concentration and storage temperatures above 24C should therefore be avoided. It is also possible that the ethyl carbamate found in wine may be the result of the breakdown of inexpensive polyurethane materials used in wine production or from the use of ethyl carbamate as a solvent for pesticides in vineyards (Ingledew et al, 1987). Experiments done by Riffkin et al (1989) indicated that the ethyl carbamate precursor in whisky distillation might be a copper-peptide or -protein complex.

Several precautions to limit the level of ethyl carbamate in wine by means of viticultural and enological practices have been previously discussed (Augustyn, 1997; Crook, 1997). Subsequently researchers in South Africa also started to address this problem either by determining the extent of the threat in SA wines or by finding a way to prevent the development of ethyl carbamate. With this article, I would like to focus on the prevention of ethyl carbamate formation with tools provided by molecular biology. The section on the heterologous expression of the L fermentum urease in the wine yeast summarises part of the research done by Ancha Zietsman for her MSc Agric degree at the Department of Microbiology, University of Stellenbosch. This study was partially funded by Winetech.

Preventing Ethyl Carbamate Formation in Wine using Molecular Biology

Disruption of the Arginase Gene. In S cerevisiae, urea is formed during the breakdown of l-arginine by the arginase enzyme encoded by the CAR1 gene (Sumrada and Cooper, 1982). Kitamoto and co-workers disrupted the two copies of this gene in the sake yeast to minimise the production of urea (Kitamoto et al, 1991, Suizu et al, 1990). The resulting mutant was used to brew sake that contained no urea or ethyl carbamate.

Removal of Urea with the Urease Enzyme. Since urea is considered by many as the main precursor of ethyl carbamate in wine and sake, the use of the urease enzyme to limit the urea levels in alcoholic beverages has been considered for many years. However, the pH conditions in wine and sake are acidic and most known ureases are inactivated at these pH levels. The Japanese sake brewers were inspired in their search for an acid urease producer and isolated the lactic acid bacterium Lactobacillus fermentum from the ileum-caecum contents of rats (Suzuki et al, 1979). Dead cells of L fermentum (Yoshizawa and Takahashi, 1988), as well as a crude cell extract (Kobashi et al, 1988), were tested for the ability to degrade urea in sake wine. The addition of 5 mg (40 IU/mg protein) of acid urease from crude cell extracts to unrefined Japanese sake containing 20% ethanol and 35 ppm urea, reduced the urea concentration to undetectable levels (1 ppm in 2 days at 15C or in 15 h at 30C).

This remarkable result was closely followed by Ough and Trioli (1988) who tested the effect of dead cells of L fermentum on the urea levels in wine. Higher concentrations of the enzyme were required to show a similar effect in wine as in sake. This was due to the lower pH of wine (the enzyme was much more effective at pH 4 than at pH 3) and the urease inhibitors that are present in wine. Ough and Trioli (1988) also established that citrulline was not removed by the urease and could still contribute to the production of ethyl carbamate. Urease activity against six other known ethyl carbamate precursors, namely N-carbamyl arginine, N-carbamyl aspartate, N-carbamyl glutamate, N-carbamyl asparagine, N-carbamyl alanine and N-carbamyl isobutyrate was also tested, but the enzyme was unable to reduce the levels of any of these compounds.

The urease enzyme was also used in reducing urea levels in a sherry base (Kodama and Yotsuzuka, 1996). Sherry undergoes a baking step at 30 – 60C for 5 to 6 weeks after fermentation and fortification. Although the levels of ethyl carbamate formed during the baking process was drastically reduced by the urease enzyme, it could not completely prevent ethyl carbamate production since ethyl carbamate precursors other than urea were still present.

Heterologous Expression of the L fermentum Urease Enzyme in Wine Yeast. Although the use of the urease enzyme in the form of dead L fermentum cells was effective in reducing the levels of urea, and therefore ethyl carbamate, it is an adverse practise to add bacterial cells to a food product. Purified urease can be added to the wine, but this is an expensive practise and requires an additional step in the wine making process. An alternative approach was to develop a wine yeast that could prevent the formation of ethyl carbamate by degrading the urea produced during fermentation. Successful expression of a heterologous urease in the wine yeast S cerevisiae, which does not contain a native urease enzyme, could enable the yeast to degrade urea to ammonia and carbon dioxide. If a suitable acid urease could be expressed, this reaction can take place at the low pH conditions associated with wine fermentation.

In this study, the lactic acid bacterium L fermentum was chosen as a source of acid urease genes. The urease enzyme of L fermentum is encoded by three contiguous open reading frames (ORF), alpha (1721 bp), beta (374 bp) and gamma (302 bp), in the urease operon (Mobley et al, 1995). This differs from eukaryotes where there is a single ORF encoding the enzyme (Figure 2) .

Since the ultimate goal of this study was to express the L fermentum enzyme in an eukaryotic yeast system, a single ORF was created by combining the three ORF’s from the bacterial urease resulting in plasmid pAV16 (Figure 3) . When comparing the urease gene sequences of L fermentum with the only eukaryotic urease gene sequence known at the time, i e that of jack bean, a very high degree of homology was observed (Figure 2) . The only major difference occurred in the two linker sequences of the jack bean gene (78 and 33 bp, respectively) that separate the three stretches that are homologous to the g, b and a subunits of L fermentum. A second construct, pAV72, was therefore designed to carry a combination of the structural genes (gamma, beta and alpha) of L fermentum and the linker sequences of the jack bean urease gene (Figure 3) . The purpose of these linker sequences was to mimic the eukaryotic model and create adequate space for the recombinant protein subunits to fold in the correct three-dimensional structure.

It has been shown that to obtain urease activity from recombinant urease genes in a host organism, one needs to express the structural genes as well as the accessory genes (Lee et al, 1992). These accessory genes have very specific functions in the incorporation of a nickel ion into the apoprotein. Although we intended to express the recombinant urease in S cerevisiae, preliminary assays were performed in Schizosaccharomyces pombe strain 603. This strain produces its own urease (specific activity: 700 to 800 mmol urea.min-1.mg protein-1 [Lubbers et al, 1996]) and will therefore contain the accessory genes to incorporate nickel ions into its native enzyme, and probably also into the recombinant enzyme. This would also allow us to evaluate the expression of the recombinant L fermentum urease genes before further work on the cloning of the accessory urease genes was undertaken. An increased urease activity in the transformed S pombe strain would indicate expression of the recombinant genes from the constitutive PGK1 promoter.

Both constructs were successfully expressed under control of the S cerevisiae PGK1 promoter and terminator signals in the yeast S pombe. Total protein extracts were obtained from the yeast transformants and compared with the extracts from the untransformed yeast and L fermentum strains for their ability to convert urea to ammonia at different pH levels. Since all the structural sections of the recombinant gene originated from L fermentum, we expected to see an acidic pH optimum for the recombinant enzyme. However, the S pombe extracts showed enhanced urease activity for the transformants only at pH 7 and the recombinant protein was highly unstable and was quickly degraded, which made the enzyme activity extremely difficult to quantify. Protein stability and activity may be improved by using a yeast host strain that is protease-deficient or by using purified recombinant protein in the enzyme assay.

This approach was only the first small step taken to resolve the problem associated with ethyl carbamate using molecular biology. For future research, the stability of the recombinant protein has to be improved, especially at low pH conditions. Furthermore, the accessory genes from L fermentum need to be isolated, cloned and expressed along with the structural genes in laboratory strains of S cerevisiae. Finally, commercial yeast strains have to be transformed with these expression cassettes to obtain stable integration into the yeast genome.

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The Authors:

Ancha Zietsman, Marinda Viljoen, Hennie van Vuuren, Department of Microbiology, University of Stellenbosch

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