Purification and characterization of a novel lipolytic enzyme from Aspergillus oryzae

Abstract

Abstract Aspergillus oryzae produced at least two kinds of extracellular lipolytic enzymes when cultured in a submerged culture medium containing 3% soybean oil. The enzymes, L1 and L2, exhibited higher substrate specificity toward dimercaptobutyrate and olive oil, respectively. The lipolytic enzyme (L1) was purified from the culture filtrate to homogeneity by ammonium sulfate and acetone precipitation, diethylaminoethyl (DEAE)-cellulose column chromatography, palmitoylated-gauze column chromatography, DEAE-Sephadex A-50 column chromatography and gel filtration, and was a monomeric protein with a molecular mass of 24 kDa estimated by gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed little preference for triacylglycerols, and also exhibited high activities on ethyl esters of various fatty acids. The enzyme cleaved all the ester bonds of triolein. The optimum pH values when olive oil and tributyrin were used as substrates were 7.5 and 10.0, respectively. One fragment derived from the enzyme cleaved off by cyanogen bromide had an N-terminal sequence of Asn-Gly-Ala-Ile-Lys-Arg-Leu-Ser-Ala-Asp-Val-Gln-Asp-Lys-Ile-Lys-Gly-Val-Val. Enzymatic properties and amino acid sequences of the lipolytic enzyme (L1) from A. oryzae and cutinases from other fungi are compared and discussed.