The hydrolysis of linalyl acetate and α-terpinyl acetate by yeasts

English: Hydrolysis of esters by means of hydrolases such as proteases (Jones and Beck, 1976), lipases (Santiello et al., 1993) and esterases (Boland et al., 1991) has become a well established method for the resolution of racemic mixtures. The first aim of the present study was to screen the yeast culture collection of the University of the Orange Free State for yeast isolates which can be used for the enantioselective hydrolysis of rac-linalyl acetate and rac-α-terpinyl acetate, which are tertiary alcohol ester terpenes, respectively. We screened 74 yeast strains from 17 genera as well as 29 unclassified isolates. Approximately 16% of the strains screened contained tertiary alcohol hydrolase activity. Whole cell experiments, enzyme purification and characterisation were attempted on one of the hydrolases of interest obtained from Trichosporon sp. UOFS Y-0117. Whole cell experiments on reported optimal hydrolase activity in the presence of 1% maltose in a defined media (YNB). The effect of various co-solvents was also documented with a low concentration of ethanol (2.4% v/v) producing superior hydrolase activity. No toxicity, to the microbe, was observed by rac-linalyl acetate (up to 200mM) due to the cell membrane present. The use of digitonin proved that substrate transport across the cell membrane is not a reaction rate determining step. The re-usability experiment showed a significant decrease in hydrolase activity (ca 30% after 1 cycle) as the same batch of cells was exposed to substrate and product. The results also show an optimal pH of 7.5 and temperature of 30°C which coincides with physiological conditions and literature. Protein purification was attempted on a cell free extract once we determined that the hydrolase was intracellular. Small scale evaluations of different chromatographic resins ranging from ion exchange, hydrophobic interaction and affinity chromatography followed. Large scale experiments with gel filtration resins were also attempted. Purification steps were largely unsuccessful and we decided to continue with a DEAE fraction which produced a superior yield (244%). Characterisation experiments, using the DEAE active fraction, followed in which we explored the effect of rac-linalyl acetate concentrations. Enzyme inhibition and protein denaturation at low rac-linalyl acetate concentrations (detected at ca 65-100mM) is significant compared to whole cells (not detected at 200mM). This hydrolase is also an esterase. Specific amino acid modification reagents results indicate the presence of a serine and histidine amino acid present in the catalytic centre i.e. the hydrolase belongs to the serine hydrolase family. A metal chelating reagent EDTA and various metal cations had no effect on hydrolase activity. pH-stability experiments indicate a pH of 7.5 to be optimal for the retention of hydrolase activity in whole cells and crude enzyme preparation. Thermostability experiments show whole cells are four times more stable than the crude enzyme preparation at 4°C. The energy of inactivation required for activity loss is lower in whole cells (47.43kJ) compared to crude enzyme preparation (91.77kJ). The probability for an event to occur which causes inactivation is relatively low (1.8075 events/h). The converse applies to the crude- enzyme preparation (1.17514 events/h). Thus the crude enzyme is 6x108 less stable than the hydrolase present in the whole cell.