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

[摘要] English: Hydrolysis of esters by means of hydrolases such as proteases (Jones andBeck, 1976), lipases (Santiello et al., 1993) and esterases (Boland et al.,1991) has become a well established method for the resolution of racemicmixtures.The first aim of the present study was to screen the yeast culture collection ofthe University of the Orange Free State for yeast isolates which can be usedfor the enantioselective hydrolysis of rac-linalyl acetate and rac-α-terpinylacetate, which are tertiary alcohol ester terpenes, respectively. We screened74 yeast strains from 17 genera as well as 29 unclassified isolates.Approximately 16% of the strains screened contained tertiary alcoholhydrolase activity. Whole cell experiments, enzyme purification andcharacterisation were attempted on one of the hydrolases of interest obtainedfrom Trichosporon sp. UOFS Y-0117.Whole cell experiments on reported optimal hydrolase activity in the presenceof 1% maltose in a defined media (YNB). The effect of various co-solventswas also documented with a low concentration of ethanol (2.4% v/v)producing superior hydrolase activity. No toxicity, to the microbe, wasobserved by rac-linalyl acetate (up to 200mM) due to the cell membranepresent. The use of digitonin proved that substrate transport across the cellmembrane is not a reaction rate determining step. The re-usabilityexperiment showed a significant decrease in hydrolase activity (ca 30% after1 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 whichcoincides with physiological conditions and literature.Protein purification was attempted on a cell free extract once we determinedthat the hydrolase was intracellular. Small scale evaluations of differentchromatographic resins ranging from ion exchange, hydrophobic interactionand affinity chromatography followed. Large scale experiments with gelfiltration resins were also attempted. Purification steps were largelyunsuccessful and we decided to continue with a DEAE fraction whichproduced a superior yield (244%).Characterisation experiments, using the DEAE active fraction, followed inwhich we explored the effect of rac-linalyl acetate concentrations. Enzymeinhibition and protein denaturation at low rac-linalyl acetate concentrations(detected at ca 65-100mM) is significant compared to whole cells (notdetected at 200mM). This hydrolase is also an esterase. Specific amino acidmodification reagents results indicate the presence of a serine and histidineamino acid present in the catalytic centre i.e. the hydrolase belongs to theserine hydrolase family. A metal chelating reagent EDTA and various metalcations had no effect on hydrolase activity. pH-stability experiments indicate apH of 7.5 to be optimal for the retention of hydrolase activity in whole cells andcrude enzyme preparation. Thermostability experiments show whole cells arefour times more stable than the crude enzyme preparation at 4°C. Theenergy of inactivation required for activity loss is lower in whole cells (47.43kJ)compared to crude enzyme preparation (91.77kJ). The probability for anevent 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 thewhole cell.

[发布机构] University of the Free State