PURIFICATION AND PROPERTIES OF CHOLESTEROL ESTERASE FROM PSEUDOMONAS-MENDOCINA-3121

Cholesterol esterase was isolated from Pseudomonas mendocina 3121. The enzyme was purified from the culture filtrate by a procedure includin g adsorption on of contaminating proteins on hydroxyapatite gel, ammon ium sulfate precipitation, isoelectric precipitation (pH 5.4), and chr omatography on Toyopearl HW-55F. The specific activity Of the purified preparation was of 31 U/mg. The molecular weight of enzyme is 62 or 3 0 kD according to gel chromatography and electrophoresis in the presen ce of SDS, respectively. The pH optimum of the enzyme activity is of 7 .3. The enzyme is stable over the pH range from 5.0 to 10.0 at tempera tures up to 50 degrees C for 30 min. The temperature optimum for enzym e activity is 55 degrees C; the pI is 5.95. The cholesterol esterase d oes not have strict substrate specificity. The K-m values for linolena te, linoleate, oleate, and palmitate were 2.0, 2.5, 4.5, and 30.7 mM, respectively. Cholesterol esterase,from Pseudomonas mendocina 3121 is inhibited by the heavy metal ions: Ag+, Zn2+, Cu2+; the corresponding inhibition constants are 0.01, 0.26, and 21 nM, respectively. The amin o acid composition of the cholesterol esterase was determined.