BIOCATALYSIS OF CHLOROPHYLLASE FROM PHAEODACTYLUM TRICORNUTM IN MICELLAR TERNARY-SYSTEM CONTAINING SPANS

The hydrolytic activity of a partially purified chlorophyllase, obtain ed from alga Phaeodactylum tricornutum, was dramatically increased in a micellar ternary system, hexane/Tris-HCl buffer/surfactant, using so rbitan surfactants of different hydrophobic chains, including sorbitan -monotaurate (Span 20), -monopalmitate (Span 40), -monostearate (Span 60), -monooleate (Span 80), and -trioleate (Span 85). The optimum conc entrations of Spans 20, 40, 60, 80, and 85, required for the hydrolyti c activity of chlorophyllase, were 15, 15, 25, 50, and 75 mu M, respec tively. The results demonstrated that the maximum partitioning, at the interface, for both enzyme and substrate was obtained with Span 85. T he V-max value for the hydrolytic activity of chlorophyllase in the he xane/aqueous medium containing Span 85 was about 288-times higher than that obtained in the absence of the surfactant. The addition of 1.0% alcohols, with different carbon chains, to the micellar system inhibit ed the enzyme activity by 25 to 100%; the degree of inhibition increas ed as the carbon-chain length of alcohols increased. The optimum value s of pH, enzyme content, incubation temperature and shaker speed, requ ired for the maximum hydrolytic activity, were 8.0, 0.5 mu g protein/m l, 35 degrees C and 300 rpm, respectively. The stability of chlorophyl lase activity in the enzymatic extracts stored at 4, 25 and 35 degrees C and shaken in a mixture of buffer solution and hexane (70:30, v/v) was temperature dependent.