Faculty of Food Technology and Biotechnology, University of Zagreb
Abstract
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⋅10^7 to 1⋅10^8 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.Piruvat dekarboksilaza (EC 4.1.1.1) jedan je od ključnih enzima u fermentativnom metabolizmu kvasca. To je prvi enzim koji u anaerobnim uvjetima dovodi do dekarboksilacije piruvata i nastanka acetaldehida. Svrha je ovog istraživanja bila razviti prikladnu metodu procjene aktivnosti piruvat dekarboksilaze različitih sojeva industrijskoga kvasca in situ. Sojevi vrsta Saccharomyces i Debaryomyces uzgojeni su u fermentacijskom mediju s 12 % glukoze. Procijenjena je aktivnost enzima u otopini stanica obrađenih digitoninom (koji povećava propusnost stanične membrane), uz dodatak natrijeva piruvata, pri temperaturi od 30 C. Razgradni metaboliti piruvata određeni su plinskom kromatografijom. Za procjenu optimalne aktivnosti piruvat dekarboksilaze in situ praćeni su razni parametri: tip i koncentracija supstrata, minimalna efektivna koncentracija digitonina, gustoća stanica, vrijeme reakcije i učinak pirazola (inhibitora alkohol dehidrogenaze). U rasponu koncentracije stanica kvasca od 1·10^7 do 1·10^8/mL zamijećena je linearna korelacija proizvedenog acetaldehida i gustoće stanica. Ustanovljeno je da je piruvat jedini specifični supstrat za enzim piruvat dekarboksilazu, te da je u prisutnosti 0,05 M natrijeva piruvata i 0,05 % digitonina enzimska reakcija imala linearni tijek prvih 20 minuta inkubacije. Tijekom inkubacije nije nastao etanol, pa je zaključeno da pirazol nije potreban u procjeni aktivnosti piruvat dekarboksilaze
