The relation between chlorophyll content and the hydrolytic activity of chlorophyllase in Chlorella protothecoides was examined. An increase in the activity was parallel to that in chlorophyll content during the development of green colouration, or greening course, in the bleached cells. The activity sharply declined and a parallel disappearance of chlorophyll was also found during bleaching of the green cells. A partially purified water-soluble preparation of chlorophyllase was obtained by n-butanol treatment and fractionation with cold acetone. It showed high activity and hydrolyzed 2 mg chlorophyll a per hr per mg protein. For separation and identification of the pigments concerned in the chlorophyllase reaction, a new solvent system of paper chromatography was introduced. When methyl chlorophyllide a and phytol were incubated with the enzyme, two products were formed. By comparison with the Rf values of isolated pure substances, one was identified as chorophyll a and the other as chlorophyllide a. This enzyme did not catalyze the phytylation of free chlorophyllide a, but it had the ability to attach phytol to methyl chlorophyllide a. The final step in the biosynthesis of chlorophyll a is briefly discussed.
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Chiba et al. (1967) studied this question.