Simon A. Schmidt

Aspartic Acid Protease from Botrytis cinerea Removes Haze-Forming Proteins during White Winemaking

White wines suffer from heat-induced protein hazes during transport and storage unless the proteins are removed prior to bottling. Bentonite fining is by far the most commonly used method, but it is inefficient and creates several other process challenges. An alternative to bentonite is the enzymatic removal of haze-forming grape pathogenesis-related proteins using added proteases. The major problem with this approach is that grape pathogenesis-related proteins are highly protease resistant unless they are heat denatured in combination with enzymatic treatment. This paper demonstrates that the protease BcAP8, from the grape fungal pathogen Botrytis cinerea , is capable of degrading chitinase, a major class of haze-forming proteins, without heat denaturation. Because BcAP8 effectively removes haze-forming proteins under normal winemaking conditions, it could potentially benefit winemakers by reducing bentonite requirements.

Epidermal Growth Factor Inhibits Gap Junctional Communication and Stimulates Serine-Phosphorylation of Connexin43 in WB Cells by a Protein Kinase C-Independent Mechanism

Epidermal growth factor (EGF) stimulated the phosphorylation of connexin43 (Cx43) in WB cells as evidenced by the formation of multiple immunoreactive Cx43 proteins of higher molecular mass which were abolished by treatment with alkaline phosphatase. Phosphorylation of Cx43 occurred within 10 min of EGF stimulation, was sustained for 1 h, and was associated with almost complete inhibition of gap junctional communication in these cells. EGF-induced phosphorylation and communication inhibition were retained in cells pretreated with phorbol 12-myristate 13-acetate (PMA) to deplete protein kinase C. These results show that the EGF inhibition of communication is tightly linked to protein kinase C-independent phosphorylation of Cx43. Further, Cx43 phosphorylated in the presence of EGF did not react with phosphotyrosine antibodies and in 32Pi incorporation experiments was shown to contain only phosphoserine indicating that the tyrosine kinase activity of the EGF receptor was not directly involved.

Hpf2 Glycan Structure Is Critical for Protection against Protein Haze Formation in White Wine

Grape-derived proteins can form haze in wine. Some cell-wall glycoproteins of Saccharomyces cerevisiae are capable of reducing protein haze formation. The basis of their haze protective activity is not yet understood. One of the S. cerevisiae cell-wall proteins, Hpf2, was produced in Pichia pastoris . An altered glycan structure in the P. pastoris -produced protein was associated with decreased solubility in water and reduced capacity to mitigate haze formation compared to native Hpf2 protein from S. cerevisiae. alpha-1,2-Linked mannose in the glycan chain was shown to be required for haze protective activity using a series of S. cerevisiae deletion mutants (mnn1-Delta, mnn2-Delta, mnn4-Delta, and mnn5-Delta), defective in different aspects of glycan processing. The effect of media additives phthalate, casamino acids, and yeast nitrogen base on Hpf2 production in P. pastoris were also evaluated. Casamino acids were shown to suppress Hpf2 production in P. pastoris .

The M flax rust resistance pre-mRNA is alternatively spliced and contains a complex upstream untranslated region

Alternative splicing is an important step in controlling gene expression and has been shown to occur for a number of plant disease resistance (R) genes. The specific biological role of alternatively spliced transcripts from most R genes is unknown, yet in two cases it is clear that functional disease resistance cannot be activated without them. We report 12 splice isoforms of the M flax rust resistance gene, a TIR–NBS–LRR class of R gene. Collectively, these isoforms are predicted to encode at least nine different polypeptide products, only one of which is a full length peptide believed to confer functional M gene-specific disease resistance. An additional intron to that previously described was found in the 5′ untranslated region. Splicing of this leader intron removes an upstream ORF (μORF) sequence. In some transcripts the leader intron is retained and in this case we predict negligible translation initiation of the full length M gene-encoding ORF. The majority of the alternatively spliced isoforms of M would encode truncated TIR and TIR–NBS containing proteins. Although the role of alternative splicing and the existence and function of the products they encode is still unclear, the complexities of the splicing profile, and the 5′ UTR of the M gene, are likely to serve in mechanisms to regulate R protein levels.

Not all wine yeast are equal

It may come as a surprise to learn that there are over 200 commercial strains of Saccharomyces cerevisiae available for winemakers to work their magic on grape juice. Why so many? Surely one or two reliable workhorse strains should suffice; after all, don?t they just make ethanol from sugar? The answer to this is an emphatic no; the more we look at the role(s) of yeast in winemaking, the more we are learning about their influences on appearance, aroma, flavour, mouthfeel and final ethanol concentration. And different yeast are more or less robust and efficient in converting the hostile environment of grape juice into wine. Indeed, not all wine yeasts are equal.