Purification and characterization of phospholipase B from Candida utilis.
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.
Laboratory of Food Biochemistry, Department of Bioresources, Faculty of Agriculture, Ehime University, Japan.
Shuji
S
Fujino
Daigo
D
Akiyama
Satoko
S
Akaboshi
Tomonari
T
Fujita
Yasuo
Y
Watanabe
Youichi
Y
Tamai
D000217:Acyltransferases / Q000378:metabolism; D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D002412:Cations / Q000737:chemistry; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008245:Lysophospholipase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008670:Metals / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D002412:Cations; D008670:Metals; D000217:Acyltransferases; D008245:Lysophospholipase