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The biocatalysis of a crude chlorophyllase extract, obtained from the biomass culture of Phaeodactylum tricornutum, in neat organic solvent media was investigated. The addition of selected excipients, including crown ether (enzyme:crown ether, 135:1--2.7:1, w/w), dextran (enzyme:dextran, 1:2--1:0.25, w/w), Span 40 and Span 60 (enzyme:Span, 1:2, w/w) and sodium bis (2-ethylhexyl) sulfosuccinate (enzyme:AOT, 1:12.6, w/w), to the crude solid enzyme preparation decreased the chlorophyllase activity. The effects of selected parameters, including solvent hydrophobicity (Log P, 2.40--4.45), initial water activity (aw, 0.44--0.97), agitation speed (0--200 rpm), reaction temperature (25--45°C) and enzyme concentration (1.67--5.3 mg solid enzyme/mL) on chlorophyllase activity, were investigated using a crude solid enzyme. The experimental findings showed that the highest chlorophyllase specific activity of 362.4 nmol hydrolyzed chlorophyll/g solid enzyme/min and bioconversion yield of 90.7% were obtained with the hexane/octanone mixture (98.7:1.3, v/v), aw of 0.90, agitation speed of 200 rpm, reaction temperature of 35°C and enzyme concentration of 3.33 mg solid enzyme/mL.

A partially purified chlorophyllase, obtained from 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 $ mu$M of Span 85 or 1 $ mu$M 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 $ mu$g protein/ml, 60 min and 27.5$ sp circ$C, respectively. The enzyme was assayed for its hydrolytic activity in the most appropriate ternary system containing Span 85 with purified pheophytin, as well as chlorophyll derivatives, as substrates. Moreover, the values of $V sb{ rm max}/K sb{ rm m}$ ratio for chlorophyllase, using the partially purified pheophytin as substrate, in ternary systems with Span 85 and Tween 80 as surfactants, were 0.15 and 0.08, respectively; however, the value of $V sb{ rm max}/K sb{ rm m}$ ratio for the enzyme, in the ternary system with Span 85, using purified pheophytin as substrate was 0.07. The addition of optimized amounts of individual membrane lipids, L-$ alpha$-phosphatidylcholine, L-$ alpha$-phosphatidyl-DL-glycerol and $ beta$-carotene increased the hydrolytic activity of chlorophyllase, using partially purified pheophytin as substrate, by 50, 36 and 10%, respectively, for Span 85, and 30, 48 and 15%, respectively, for Tween 80; in addition, these lipids increased the enzyme activity by 6, 23 and 31%, respectively, in the Span 85 media, using purified pheophytin as substrate. Phytol showed a competitive inhibitory effect on chlorophyllase activity in both Span and Tween systems containing partially purified pheophytin substrate; however, phytol had an uncompetitive inhibitory effect on the enzyme activity in the S

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Thesis (Ph.D.)--Tufts University, 2004. / Adviser: Krishna Kumar. Submitted to the Dept. of Chemistry. Includes bibliographical references (leaves 190-197). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xix, 140 p. : ill. Includes abstract. Includes bibliographical references (p. 133-140).