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Biocatalysis of Chlorophyllase in Ternary Micellar Systems Using Pheophytins as Substrates
Author(s) -
Samaha Hiba,
Kermasha Selim
Publication year - 1997
Publication title -
journal of chemical technology and biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 117
eISSN - 1097-4660
pISSN - 0268-2575
DOI - 10.1002/(sici)1097-4660(199703)68:3<315::aid-jctb654>3.0.co;2-9
Subject(s) - pheophytin , chemistry , chromatography , hydrolysis , phaeodactylum tricornutum , phytol , biochemistry , botany , biology , photosynthesis , photosystem ii , diatom
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. © 1997 SCI.