Abstract
Cholesterol ester hydrolase (EC 3. 1. 1. 13) was purified about 10 times from the culture supernatant of Pseudomonas fluorescens ATCC 21156 by a procedure involving fractionations with acetone and ammonium sulfate, chromatography on hydroxylapatite, gel filtration on Sephadex G-100, and separation into two isoenzymes, I and II, by isoelectric focusing. The purified enzyme from the Sephadex G-100 fraction sedimented as a single peak on ultracentrifugation. The isoenzymes I and II showed isoelectric points of pH 3.8 and 4.9, respectively. They both had an apparent molecular weight of about 129, 000, hydrolyzed preferentially long-chain fatty acid esters of cholesterol, and exhibited a pH optimum at 7.3 with cholesterol linoleate as substrate. The Km values of I and II for cholesterol linoleate were 3.86×10-5 M and 1.43×10-4 M, respectively. The cholesterol esterase activity was remarkably stimulated when Triton X-100 was used to prepare the miscellar aqueous substrate, and further increase in activity was demonstrated by the addition of cholic acid to the miscellar system.
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