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Pyruvate Decarboxylase Activity Assay in situ of Different Industrial Yeast Strains 

Joanna Berłowska*, Dorota Kręgiel and Wojciech Ambroziak


Institute of Fermentation Technology and Microbiology, Technical University of Lodz, Wolczanska 171/173, PL-90-924 Lodz, Poland

Article history:

Received November 21, 2007
Accepted July 28, 2008

Key words:

enzymatic activity in situ, pyruvate decarboxylase, yeasts

Summary:

Cytoplasmic pyruvate decarboxylase (PDC, EC 4.1.1.1) is one of the key enzymes of yeast fermentative metabolism. PDC is the first enzyme which, under anaerobic conditions, leads to decarboxylation of pyruvate with acetaldehyde as the end product. The aim of this study is to develop a suitable method for PDC activity assay in situ for different industrial yeast strains. Saccharomyces sp. and Debaryomyces sp. yeast strains grew in fermentative medium with 12 % of glucose. Enzymatic assay was conducted in cell suspension treated with digitonin as permeabilisation agent, and with sodium pyruvate as a substrate, at temperature of 30 °C. Metabolites of PDC pathway were detected using gas chromatographic (GC) technique. Various parameters like type and molar concentration of the substrate, minimal effective mass fraction of digitonin, cell concentration, reaction time and effect of pyrazole (alcohol dehydrogenase inhibitor) were monitored to optimize PDC enzymatic assay in situ. In the concentration range of yeast cells from 1⋅107 to 1⋅108 per mL, linear correlation between the produced acetaldehyde and cell density was noticed. Only pyruvate was the specific substrate for pyruvate decarboxylase. In the presence of 0.05 M sodium pyruvate and 0.05 % digitonin, the enzymatic reaction was linear up to 20 min of the assay. During incubation, there was no formation of ethanol and, therefore, pyrazole was not necessary for the assay.

 


*Corresponding author:           joanna_berlowska@vp.pl
                                               ++48 42 6313 476
                                               ++48 42 6365 976

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