Extracellular acid proteases of wine microorganisms : gene identification, activity characterization and impact on wine

[摘要] ENGLISH ABSTRACT:Non-Saccharomyces yeasts of oenological origin have previously been associated with spoilage orregarded as undesired yeasts in wine. However, these yeasts have recently come under investigation fortheir positive contribution towards wine aroma especially when used in sequential or co-inoculatedfermentations with Saccharomyces cerevisiae. These yeasts are also known to secrete a number ofenzymes that could be applicable in wine biotechnology. Amongst these enzymes are aspartic proteases.The secreted proteases from some non-Saccharomyces yeast may play a role in protein haze reduction,as demonstrated by some authors, while simultaneously increasing the assimilable nitrogen content ofthe wine for the utilization and growth of fermentative microorganisms. Moreover, the proteases may havean indirect effect on wine aroma by liberating amino acids that serve as aroma precursors. Althoughmany screenings have been performed detecting protease activity in non-Saccharomyces yeasts, noattempts have been made to characterize these enzymes. This study set out to isolate and characterizegenes encoding extracellular aspartic proteases from non-Saccharomyces yeasts.An enzymatic activity screening of a collection of 308 Saccharomyces and non-Saccharomyces yeasts,isolated from grape must, was performed. The aspartic protease-encoding genes of two non-Saccharomyces yeasts, which showed strong extracellular proteolytic activity on plate assays, wereisolated and characterized by in silico analysis. The genes were isolated by employing degenerate andinverse PCR. One gene was isolated from Metschnikowia pulcherrima IWBT Y1123 and named MpAPr1.The other putative gene was isolated from Candida apicola IWBT Y1384 and named CaAPr1. TheMpAPr1 gene is 1137 bp long, encoding a 378 amino acid putative protein with a predicted molecularweight of 40.1 kDa. The CaAPr1 putative gene is 1101 bp long and encodes a 367 amino acid putativeprotein with a predicted molecular weight of 39 kDa. These features are typical of extracellular asparticproteases. The deduced protein sequences showed less than 40% homology to other yeast extracellularaspartic proteases. By heterologous expression of MpAPr1 in S. cerevisiae, it was confirmed that thegene encodes an extracellular acid protease. The expression of MpAPr1 was shown to be induced inmedia containing proteins as sole nitrogen source and repressed when a preferred nitrogen source wasavailable. The gene was expressed in the presence of casein, bovine serum albumin (BSA) and grapejuice proteins and repressed in the presence of ammonium sulphate. Expression was most induced in thepresence of grape juice proteins, which was expected since these proteins are present in the naturalhabitat of the yeast. A genetic screening confirmed the presence of the MpAPr1 gene in 12 otherM. pulcherrima strains isolated from grape juice. The extracellular protease activity of the strains was alsovisualized on plates. As far as we know, this is the first report on the genetic characterization of secretedaspartic proteases from non-Saccharomyces yeasts isolated from grape must and provides thegroundwork for further investigations.

[发布机构] Stellenbosch University