Takó Miklós, KotogÁn Alexandra, Papp TamÁs, Kadaikunnan Shine, Alharbi Naiyf S, VÁgvölgyi Csaba
Department of Microbiology, Faculty of Science and Informatics, University of Szeged, H-6726 Szeged, Közép fasor 52, Hungary.
HAS-USZ "Momentum" Fungal Pathogenicity Mechanisms Research Group, University of Szeged, H-6726 Szeged, Közép fasor 52, Hungary.
J Microbiol Biotechnol. 2017 Feb 28;27(2):277-288. doi: 10.4014/jmb.1608.08005.
NRRL 5282 and NRRL 1526 can produce lipases with high synthetic activities in wheat bran-based solid-state culture. In this study, the purification and biochemical characterization of the lipolytic activities of these lipases are presented. SDS-PAGE indicated a molecular mass of about 55 and 35 kDa for the purified and enzymes, respectively. -Nitrophenyl palmitate (NPP) hydrolysis was maximal at 40°C and pH 7.0 for the lipase, and at 30°C and pH 5.2 for the enzyme. The enzymes showed almost equal affinity to NPP, but the of the lipase was about 1.13 times higher than that determined for using the same substrate. For both enzymes, a dramatic loss of activity was observed in the presence of 5 mM Hg, Zn, or Mn, 10 mM -bromosuccinimide or sodium dodecyl sulfate, and 5-10% (v/v) of hexanol or butanol. At the same time, they proved to be extraordinarily stable in the presence of -hexane, cyclohexane, -heptane, and isooctane. Moreover, isopentanol up to 10% (v/v) and propionic acid in 1 mM concentrations increased the NPP hydrolyzing activity of lipase. Both enzymes had 1,3-regioselectivity, and efficiently hydrolyzed -nitrophenyl (NP) esters with C8-C16 acids, exhibiting maximum activity towards NP-caprylate () and pNP-dodecanoate (). The purified lipases are promising candidates for various biotechnological applications.
NRRL 5282和NRRL 1526在基于麦麸的固态培养中能够产生具有高合成活性的脂肪酶。在本研究中，展示了这些脂肪酶脂解活性的纯化及生化特性。SDS-PAGE表明，纯化后的两种酶分子量分别约为55 kDa和35 kDa。对于第一种脂肪酶，对硝基苯基棕榈酸酯（NPP）的水解在40°C和pH 7.0时达到最大值，而对于第二种酶，在30°C和pH 5.2时达到最大值。这两种酶对NPP表现出几乎相同的亲和力，但第一种脂肪酶的催化效率比使用相同底物测定的第二种酶高约1.13倍。对于这两种酶，在存在5 mM的汞、锌或锰、10 mM的N-溴代琥珀酰亚胺或十二烷基硫酸钠以及5-10%（v/v）的己醇或丁醇时，观察到活性急剧丧失。同时，它们在存在正己烷、环己烷、正庚烷和异辛烷时被证明具有极高的稳定性。此外，高达10%（v/v）的异戊醇和1 mM浓度的丙酸可提高第一种脂肪酶的NPP水解活性。两种酶都具有1,3-区域选择性，并且能有效水解含有C8-C16酸的对硝基苯基（NP）酯，对NP-辛酸酯（）和对硝基苯基十二烷酸酯（）表现出最大活性。纯化后的脂肪酶是各种生物技术应用的有前景的候选者。
