Fujino Shuji, Akiyama Daigo, Akaboshi Satoko, Fujita Tomonari, Watanabe Yasuo, Tamai Youichi
Laboratory of Food Biochemistry, Department of Bioresources, Faculty of Agriculture, Ehime University, Japan.
Biosci Biotechnol Biochem. 2006 Feb;70(2):377-86. doi: 10.1271/bbb.70.377.
Phospholipase B (PLB) from the asporogenous yeast Candida utilis was purified to homogeneity from a culture broth. The apparent molecular mass was 90-110 kDa by SDS-PAGE. The enzyme had two pH optima, one acidic (pH 3.0) and the other alkaline (pH 7.5). At acidic pH the enzyme hydrolyzed all phospholipids tested without metal ions. On the other hand, the PLB showed substrate specificity and required metal ions for alkaline activity. The cDNA sequence of the PLB was analyzed by a combination of several PCR procedures. The PLB encoded a protein consisting of 643 amino acids. The amino acid sequence contained a lipase consensus sequence (GxSxG) and catalytic arginine and aspartic acid motifs which were identified as the catalytic triad in the PLB from Kluyveromyces lactis, suggesting that the catalytic mechanism of the PLB is similar to that of cytosolic phospholipase A(2) (cPLA(2)), found in mammalian tissues.
从产朊假丝酵母的无孢子酵母中纯化出了磷脂酶B（PLB），使其达到了从培养液中纯化至同质的程度。通过SDS-PAGE分析，其表观分子量为90 - 110 kDa。该酶有两个最适pH值，一个是酸性（pH 3.0），另一个是碱性（pH 7.5）。在酸性pH条件下，该酶无需金属离子就能水解所有测试的磷脂。另一方面，PLB表现出底物特异性，碱性活性需要金属离子。通过多种PCR方法相结合对PLB的cDNA序列进行了分析。PLB编码一种由643个氨基酸组成的蛋白质。氨基酸序列包含一个脂肪酶共有序列（GxSxG）以及催化性精氨酸和天冬氨酸基序，这些在乳酸克鲁维酵母的PLB中被鉴定为催化三联体，这表明PLB的催化机制与在哺乳动物组织中发现的胞质磷脂酶A(2)（cPLA(2)）相似。
