Molecular characterization of an extracellular acid-resistant lipase produced by Rhizopus javanicus.
An extracellular lipase (triacylglycerol acylhydrolase EC 3.1.1.3), produced by the fungus Rhizopus javanicus was purified to homogeneity using an expeditious two-step isolation method. The enzyme, with a molecular mass of 36 kDa and a specific activity of 9260 microequivalent of fatty acid released per minute and mg under standard conditions, consists of three isoforms with isoelectric points of 7.8, 7.7, and 7.1, respectively. The purified lipase was digested using chemical and enzymatical procedures: CNBr cleavage, partial acid hydrolysis, and proteolytic cleavage by means of trypsin. Amino-acid sequencing of the resulting peptides indicates that the three lipases from Rhizopus javanicus, Rhizopus niveus and Rhizopus delemar are produced as identical proenzymes but processed differently. These Rhizopus lipases show 54% identity with the lipase from Rhizomucor miehei. Using the structure of the Rhizomucor miehei lipase, the molecular model of Rhizopus javanicus lipase was constructed. Both enzymes are alpha/beta type proteins with a central 8-stranded mixed beta-pleated sheet and have a remarkably similar distribution of hydrophobic amino acids at their surface. The tryptophan in the center of the helical lid covering the active site of Rhizomucor miehei lipase is mutated into an alanine, indicating that it is not essential for the proper movement of the helical lid.
Uyttenbroeck
W
W
Universiteit Antwerpen, UIA, Wilrijk, België.
Hendriks
D
D
Vriend
G
G
De Baere
I
I
Moens
L
L
Scharpé
S
S
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007527:Isoenzymes; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012233:Rhizopus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D005227:Fatty Acids; D007527:Isoenzymes; D008049:Lipase