Purification and Some Properties of Soluble Phospholipase B from Baker's Yeast (Saccharomyces cerevisiae)

Abstract

Phospholipase B from baker's yeast (Saccharomyces cerevisiae) was purified by acid treatment of the crude extract, ammonium sulfate fractionation, and column chromatographies on DEAE-Sepharose CL-6B, Sepharose 4B, and Bio-Gel HTP. The purified preparation had lysophospholipase activity and phospholipase B activity in a ratio of 16:1. The optimum pH of both activities was 3.5-4.0. The enzyme was a glycoprotein and its molecular size was somewhat heterogeneous, ranged from about 280, 000 to 420, 000 by gel nitration. Phospholipase B activity was strongly stimulated by 0.1% DOC, but lysophospholipase activity was completely inhibited by the detergent. Neither activity was stimulated by Ca²⁺ and both were inhibited by SDS, Triton X-100, and Fe³⁺. The enzyme hydrolyzed the acyl ester bonds of phosphatidylcholine sequentially, first the 2-acyl and then the 1-acyl groups. The Km values for phosphatidylcholine and lysophosphatidylcholine were 0.63 mM and 0.05 mM, respectively.