The aim of this study was to evaluate the efficiency of sonication in releasing protein from a widespread lipase-producing yeast, Yarrowia lipolytica KKP 379, and to examine the impact of ultrasound waves generated in a horn-type sonicator on the lipolytic activity of Y. lipolytica in the hydrolysis of p-nitrophenyl laurate. In this paper, we focused on a few parameters of ultrasound cell disruption, such as the time of sonication, acoustic power, storage time of the frozen yeast biomass used in sonication and the solvent used to suspend the yeast cells which were considered as the most important part in the process of obtaining a biocatalyst from Y. lipolytica for organic synthesis. The most effective additive in protein release proved to be 2% Tween 80; other ideal parameters of the process were ultrasonic power at 150 W for 15 min and 9 weeks of frozen biomass storage time. The sonication parameters, which were the best for protein release, did not seem to be the most effective for obtaining high lipolytic activity due to denaturation as an effect of cavitation.
Techniki ultradźwiękowe są od dawna stosowane w przemyśle spożywczym, przede wszystkim w procesach przetwarzania i utrwalania żywności. Celem pracy była ocena sonifikacji jako alternatywnej metody inaktywacji komórek drożdży. Dodatkowo rozważono zastosowanie sonifikacji do pozyskiwania roztworu białek wewnątrzkomórkowych. Komórki szczepu drożdży Saccharomyces cerevisiae 2200 oraz komórki świeżych drożdży piekarskich poddawano sonifikacji w głowicowym homogenizatorze ultradźwiękowym o częstotliwości 20 kHz. Zaobserwowano istotny wpływ czasu, cyklu pracy oraz mocy fali akustycznej na inaktywację komórek oraz stopień izolacji białek, które wydzielono z wydajnością 60 % ze szczepu drożdży S. cerevisiae 2200 i 43 % ze świeżych drożdży piekarskich. Dezintegracja struktury komórkowej drożdży za pomocą ultradźwięków może być dobrą laboratoryjną metodą permeabilizacji ściany komórkowej oraz izolacji białek. Liczba żywych komórek po procesie sonifikacji zmniejszyła się 100 do 1000-krotnie w porównaniu z początkowąj liczbą jtk/cm 3 , przy czym efekt ten może zostać zwielokrotniony przez połączenie działania ultradźwięków z czynnikiem termicznym.
Chemical Biology 212 yields from lignocellulosic biomass from agricultural and agro-industrial residues are dependent upon efficient hydrolysis of sugar polymers and utilization of all the available sugars including D-glucose, D-xylose, L-arabinose and other fermentable compounds. S. cerevisiae, which plays a traditional and major role in industrial bioethanol production, has several advantages due to its high ethanol productivity as well as its high ethanol tolerance. However, baker's yeast cannot hydrolyze cellulose and is not able to use pentoses, which constitute up to 20% of lignocellulosic biomass. Many studies regarding the use of S. cerevisiae in metabolic engineering for xylose utilization have been reported and several reviews have been published [Chu & Lee, 2007; Hahn-Hägerdal et al., 2007; Matsushika et al., 2009]. The first step of D-xylose metabolism is its direct isomerization to D-xylulose catalyzed by bacterial xylose isomerase XR (EC 5.3.1.5) or stepwise transformation in yeast cells, firstly to xylitol (xylose reductase XR EC 1.1.1.21) and then to D-xylulose (xylitol dehydrogenase XDH EC 1.1.1.19). After phosphorylation of D-xylulose to D-xylulose-5phosphate (xylulokinase XK EC 2.7.1.17) further metabolizm proceeds via a pentose phosphate pathway. Different strategies have been applied to engineering yeast including the introduction of initial xylose metabolism and xylose transport. However, these change the intracellular redox balance and result in over-expression of xylulokinase and further metabolism via a pentose phosphate pathway. Nevertheless they are insufficient for industrial bioprocesses mainly due to a low rate of reaction as compared with glucose fermentation [Kondo et al. 2010; Young et al., 2010].
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