Purification and Characterization of Extracellular Lipases Produced by Geotrichum sp. FO347-2

未精製鰯油を唯一の炭素源としてスクリーニングされた真菌類の中で、FO347-2株は最もリパーゼ生産性が高い株であった。本株は、同定の結果、Geotrichum sp. と推定された。また、菌体外に複数のリパーゼを分泌していたので、これらのリパーゼを精製し、酵素化学的性質を検討した。培養液の遠心上清をアセトン沈澱した後、DEAE-Toyepearl 650MとButyl-Toyopearl 650Mでカラムクロマトグラフイーを行った。その結果、前者のカラムでLipase I、IIの2つの活性画分を得、Iは更に後者のカラムでLipase A、 Bの2つの活性画分に分かれた。AとBは SDS-PAGE でそれぞれ単一なバンドであり、分子量マーカーより推定された分子量はA、Bそれぞれ、62kD, 58kDであった。また、AとBの反応至適pHは、ともに8.5であった。位置特異性(PSI)を調べた結果、Aは+47.0、Bは-25.7であり、精製鰯油を基質として脂肪酸特異性を調べた結果、AはC16:1、BはC18:1に最も高い特異性を示した。A fungus, FO347-2, produced the largest amount of lipase among the microorganisms that were isolated as microorganisms capable of assimilating sardine oil as a sole carbon source; it was identified as Geotrichum sp. As FO347-2 secreted several extracellular lipases, they were purified to examine their enzymatic properties. They were purified from the culture supernatant by ammonium sulfate fractionation, ion exchange chromatography, and hydrophobic interaction chromatography. Two peaks of lipase activity were separated as Lipase I and II by ion exchange chromatography. Further, Lipases A and B were obtained from the Lipase I fraction by hydrophobic interaction chromatography. Lipases A and B were homogeneous in SDS-PAGE and their molecular weights were 62 and 58kD, respectively. The optimum pHs of Lipases A and B were both 8.5. The positional specificity was expressed as PSI. The PSI values of Lipases A and B were +47.0 and -25.7, respectively. When the concentration rate of each fatty acid hydrolyzed by Lipases A or B in sardine oil was measured, Lipases A and B hydrolyzed the ester bonds of C16:1 and C18:1 with the highest velocity, respectively