MOLECULAR CHARACTERIZATION OF AN EXTRACELLULAR ACID-RESISTANT LIPASE PRODUCED BY RHIZOPUS-JAVANICUS

An extracellular lipase (triacylglycerol acylhydrolase EC 3.1.1.3), pr oduced by the fungus Rhizopus javanicus was purified to homogeneity us ing an expeditious two-step isolation method. The enzyme, with a molec ular mass of 36 kDa and a specific activity of 9260 microequivalent of fatty acid released per minute and mg under standard conditions, cons ists of three isoforms with isoelectric points of 7.8, 7.7, and 7.1, r espectively. The purified lipase was digested using chemical and enzym atical procedures: CNBr cleavage, partial acid hydrolysis, and proteol ytic cleavage by means of trypsin. Amino-acid sequencing of the result ing peptides indicates that the three lipases from Rhizopus javanicus, Rhizopus niveus and Rhizopus delemar are produced as identical proenz ymes but processed differently. These Rhizopus lipases show 54% identi ty with the lipase from Rhizomucor miehei. Using the structure of the Rhizomucor miehei lipase, the molecular model of Rhizopus javanicus li pase was constructed. Both enzymes are alpha/beta type proteins with a central 8-stranded mixed beta-pleated sheet and have a remarkably sim ilar distribution of hydrophobic amino acids at their surface. The try ptophan in the center of the helical lid covering the active site of R hizomucor miehei lipase is mutated into an alanine, indicating that it is not essential for the proper movement of the helical lid.