Extracellular acid proteases of wine microorganisms : gene identification, activity characterization and impact on wine
Year: 2012
Type: dissertation
Abstract: Non-Saccharomyces yeasts of oenological origin have previously been associated with spoilage or regarded as undesired yeasts in wine.However, these yeasts have recently come under investigation for their positive contribution towards wine aroma especially when used in sequential or co-inoculated fermentations with Saccharomyces cerevisiae.These yeasts are also known to secrete a number of enzymes 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 of the wine for the utilization and growth of fermentative microorganisms.Moreover, the proteases may have an indirect effect on wine aroma by liberating amino acids that serve as aroma precursors.Although many screenings have been performed detecting protease activity in non-Saccharomyces yeasts, no attempts have been made to characterize these enzymes.This study set out to isolate and characterize genes 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, were isolated and characterized by in silico analysis.The genes were isolated by employing degenerate and inverse 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.The MpAPr1 gene is 1137 bp long, encoding a 378 amino acid putative protein with a predicted molecular weight of 40.1 kDa.The CaAPr1 putative gene is 1101 bp long and encodes a 367 amino acid putative protein with a predicted molecular weight of 39 kDa.These features are typical of extracellular aspartic proteases.The deduced protein sequences showed less than 40% homology to other yeast extracellular aspartic proteases.By heterologous expression of MpAPr1 in S. cerevisiae, it was confirmed that the gene encodes an extracellular acid protease.The expression of MpAPr1 was shown to be induced in media containing proteins as sole nitrogen source and repressed when a preferred nitrogen source was available.The gene was expressed in the presence of casein, bovine serum albumin (BSA) and grape juice proteins and repressed in the presence of ammonium sulphate.Expression was most induced in the presence of grape juice proteins, which was expected since these proteins are present in the natural habitat of the yeast.A genetic screening confirmed the presence of the MpAPr1 gene in 12 other M. pulcherrima strains isolated from grape juice.The extracellular protease activity of the strains was also visualized on plates.As far as we know, this is the first report on the genetic characterization of secreted aspartic proteases from non-Saccharomyces yeasts isolated from grape must and provides the groundwork for further investigations.
Author: Vernita Jennilee Reid
Language: English
Subfield: Food Science
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W325479809