ABSTRACT
AIM: Plant materials used in the food industry contain up to five times more aromas bound to glucose (glucosides) than free, unbound aromas, making these bound aromas an unused flavouring potential. The aim of this study was to identify and purify a novel ß-glucosidase from Brettanomyces yeasts that are capable of releasing bound aromas present in various food products. METHODS AND RESULTS: We screened 428 different yeast strains for ß-glucosidase activity and are the first to sequence the whole genome of two Brettanomyces yeasts (Brettanomyces anomalus and Brettanomyces bruxellensis) with exceptionally high ß-glucosidase activity. Heterologous expression and purification of the identified B. anomalus ß-glucosidase showed that it has an optimal activity at a higher pH (5·75) and lower temperature (37°C) than commercial ß-glucosidases. Adding this B. anomalus ß-glucosidase to cherry beers and forest fruit milks resulted in increased amounts of benzyl alcohol, eugenol, linalool and methyl salicylate compared to Aspergillus niger and Almond glucosidase. CONCLUSIONS: The newly identified B. anomalus ß-glucosidase offers new possibilities for food bioflavouring. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to sequence the B. anomalus genome and to identify the ß-glucosidase-encoding genes of two Brettanomyces species, and reports a new bioflavouring enzyme.
Subject(s)
Brettanomyces/metabolism , Flavoring Agents/metabolism , Fungal Proteins/metabolism , beta-Glucosidase/metabolism , Brettanomyces/chemistry , Brettanomyces/genetics , Enzyme Stability , Fermentation , Flavoring Agents/analysis , Food Microbiology , Fungal Proteins/chemistry , Fungal Proteins/genetics , Prunus/metabolism , Prunus/microbiology , beta-Glucosidase/chemistry , beta-Glucosidase/geneticsABSTRACT
The yeast Saccharomyces cerevisiae responds to amino acid deprivation by increasing translation of the transcription factor Gcn4, which enhances expression of amino acid biosynthetic genes. Accumulation of uncharged tRNAs activates the Gcn2 protein kinase, which phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (eIF2α). The resulting downregulation of eIF2 activity causes reduction of general translation and stimulation of GCN4 translation. S. cerevisiae contains three PDK1 orthologs (encoded by PKH1, PKH2 and PKH3) that have been implicated in nutrient signaling. Using heterologously expressed proteins, we demonstrate physical interaction between Pkh1 and all three subunits of eIF2 as well as Gcn2. We confirm the interaction between Pkh1 and Gcn2 by co-immunoprecipitation in yeast cell extracts and show that Pkh1 can phosphorylate Gcn2 in vitro. However, Pkh1 inactivation did not affect eIF2α-S51 phosphorylation in vivo or GCN4 translation in response to amino acid deprivation. Hence, the physiological importance of the close interactions between Pkh1 and Gcn2 or eIF2 could depend on other conditions and/or other targets of the Gcn2/eIF2 system.
