Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as: a Rhizopus oryzae. Culture conditions were optimized and highest lipase pro duction amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitat ion, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration an d a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mas s of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. T he enzyme exhibited a single band in active polyacrylamide gel electrophore sis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase b y RP-HPLC confirmed the homogeneity of the enzyme preparation. Determinatio n of the N-terminal sequence over 19 amino acid residues showed a high homo logy with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temper ature for lipase activity was 35 degrees C and about 65% of its activity wa s retained after incubation at 45 degrees C for 30 min. The lipolytic enzym e was inhibited by Triton X100, SDS, and metal ions such as Fe3+, CU2+, Hg2 + and Fe2+. Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis o f saturated fatty acid chains (C-8-C-18) and a 1, 3-position specificity. I t showed a good stability in organic solvents and especially in long chain- fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for se lective esterification of sardine free fatty acids with hexanol as substrat e. About 76% of sardine free fatty acids were esterified after 30 h reactio n whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterifi ed fatty acids. (C) 2000 Elsevier Science Inc. All rights reserved.