Biocatalysis of chlorophyllase in a ternary micellar system containing Span 85 using purified and oxidized pheophytins as substrates

Abstract

Partially purified chlorophyllase, obtained from the alga Phaeodactylum tricornutum, was assayed for its hydrolytic activity in a ternary micellar system containing Span 85, using as substrates purified pheophytins and chlorophyll derivatives (pheophytins and pyropheophytins). The effects of enzyme content, substrate concentration, membrane lipids and phytol on chlorophyllase activity were investigated. The V max and K m values for chlorophyllase activity, using the purified pheophytin as substrate, were 13.3 nmol hydrolyzed pheophytin per mg protein per min and 206 nmol pheophytin per liter, respectively. The addition of optimized amounts of individual membrane lipids, β-carotene, l- α-phosphatidyl- dl-glycerol and l- α-phosphatidylcholine, increased the hydrolytic activity of chlorophyllase by 31, 23 and 6%, respectively, whereas a mixture of these lipids increased the enzyme activity by 27%. Phytol showed an uncompetitive inhibitory effect on chlorophyllase activity, with a K i value of 3.65 μM, using purified pheophytin as substrate. High-performance liquid chromatography, spectrophotometric and Fourier transform infrared spectroscopic analyses were used to characterize the substrates as well as the end-products of the chlorophyllase hydrolytic activity.