Biochemical Characterization of a Lipolytic Enzyme From Aspergillus oryzae That Hydrolyzes Triacylglycerol and Sterol Es
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Biochemical Characterization of a Lipolytic Enzyme From Aspergillus oryzae That Hydrolyzes Triacylglycerol and Sterol Esters Kyotaro Ichikawa 1 & Ayaka Yoshida 1 & Yoshihito Shiono 1 & Takuya Koseki 1 Received: 28 April 2020 / Accepted: 22 June 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A novel lipolytic enzyme-encoding gene, lipO745, from Aspergillus oryzae RIB40 was cloned and expressed in Pichia pastoris. Purified recombinant LipO745 (rLipO745) had a molecular mass of approximately 60 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. rLipO745 exhibited maximum activity at 40 °C and pH 7.0 and was stable at temperatures ≤ 40 °C. The substrate specificity of purified rLipO745 was analyzed using α-naphthyl esters as artificial substrates and various triacylglycerol and sterol esters as natural substrates. From among a panel of α-naphthyl esters (C2–C16), αnaphthyl butyrate (C4), with an activity of 269 ± 3.3 units/mg protein, was the optimal substrate for hydrolysis by the purified recombinant protein. The Km and kcat values of rLiO745 for the C4 substrate were 0.073 ± 0.0012 mM and 608 ± 108 s−1, respectively. The purified recombinant enzyme had considerable hydrolytic activity toward tributyrin, tripalmitin, and triolein, indicating lipase activity, and toward cholesteryl acetate, butyrate, palmitate, and oleate, indicating sterol esterase activity. Transesterification activities between tributyrin and cholesterol or between tributyrin and campesterol were also determined. Keywords Lipase . Sterol esterase . Aspergillus oryzae . Pichia pastoris
Introduction Lipases are enzymes that catalyze the hydrolysis of diverse glycerol esters. Triacylglycerol lipases (EC 3.1.1.3) hydrolyze the triglycerides to diglycerides, monoglycerides, free fatty Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-02003360-4) contains supplementary material, which is available to authorized users.
* Takuya Koseki [email protected]–u.ac.jp
1
Department of Food, Life and Environmental Sciences, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka 997-8555, Japan
Applied Biochemistry and Biotechnology
acids, and glycerol. Triacylglycerol lipases are present ubiquitously in nature, from microorganisms to plants and animals. Lipases have potential applications in food, detergents, cosmetics, pharmaceutical, textile, and paper industries among others, due to their reactions of hydrolysis and synthesis [1, 2]. Some of the most commercially important lipases belong to yeasts, such as Candida rugosa (formerly Candida cylindracea) and Candida antarctia, or filamentous fungi, such as Aspergillus, Penicillium, and Rhizopus species, Humicola lanuginosa, and Mucor miehei [3, 4]. Microbial lipases are a useful enzyme for industrial applications due to their stability, selectivity, or broad substrate specificity. Meanwhile, some lipases hydrolyze not only triglycerides but also fatty acid esters of sterols. Stero
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