Purification and characterization of lipase from endophytic fungus Alternaria alternata isolate PTM7

Abstract

Sixty five endophytic fungal isolates, were tested for extracellular lipase production. Only ten isolates were found to produce lipase activity. Fusarium oxysporum isolated PTM 7, which was isolated from Croton oblongifolius Roxb. (Plao yai leaves), gave the highest activity. Highest lipase activity PTM 7 was shown in the basal culture medium was supplemented with 1% (v/v) olive oil, 1% (w/v) peptone and 0.5% (w/v) sodium nitrate as the carbon, organic and inorganic nitrogen sources, respectively. Purification PTM 7 extracellular lipase by precipitated in 80% saturation ammonium sulfate and molecule separation by DEAE-cellulose anion exchange and Superdex-75 gel filtration chromatography increased 41.4 in time apparent homogeneity but final yield decreased to 2.21 percent. The enriched lipase showed maximum stability at pH 8.0 and 30oC, was reasonably stable up to 40°C and at pH between 8.0 to 12.0. One mM Ca²⁺ and Mg²⁺ stimulated enzyme production and especially Mn²⁺ stimulated 133%, but inhibited by Cu2+, Fe²⁺, Hg²⁺ and Zn²⁺ 1 - 10 mM. 5 and 10 mM EDTA inhibited lipase activity but stimulatory at 1 mM. The Km and Vmax values were rather low at 2.78 mM and 9.09 mol/min/mg protein, respectively, when using p-nitrophenyl palmitate as the substrate. Enzymatic transesterification, which ratio of oil : methanol mole (1 : 6) and 6 U enzyme for biodiesel production was rather low only 28.4% FAMEs yield.