Biocatalysis of Chlorophyllase in Ternary Micellar Systems Using Pheophytins as Substrates

Abstract

The partially purified chlorophyllase, obtained from the alga Phaeodactylum tricornutum, was assayed for its hydrolytic activity towards the pheophytin in ternary micellar systems of hexane/Tris-HCl/surfactant. A wide range of surfactants, sorbitans (Span 20, 40, 60, 80 and 85) and polysorbates (Tween 20, 40, 60, 80 and 85), was used. The use of either 50 μmol dm -3 of Span 85 or 1 μmol dm -3 of Tween 80 increased the hydrolytic activity of chlorophyllase by 110 and 23%, respectively. The optimum values of pH, enzyme content, incubation time and temperature for the hydrolytic activity of chlorophyllase were determined as 8.25, 8.00 μg protein cm -3 , 60 min and 27.5°C, respectively. The V max and K m values were 6.91 nmole hydrolyzed pheophytin mg -1 protein min -1 and 47.2 nmole pheophytin dm -3 , respectively, in the Span 85 medium and 10.04 and 121.00, respectively, in the Tween 80 medium. The addition of optimized amounts of individual membrane lipids, L-α-phosphatidylcholine, L-α-phosphatidyl-DL-glycerol and β-carotene increased the hydrolytic activity of chlorophyllase by 50, 36 and 10%, respectively, for Span 85 and 30, 48 and 15%, respectively, for Tween 80. Phytol showed a competitive inhibitory effect on chlorophyllase activity in both Span and Tween systems with a K i value of 15.5 and 14.3 μmol dm -3 , respectively. High-performance liquid chromatography and spectrophotometry analyses were used to characterize the end-products of chlorophyllase hydrolytic reaction.