Tavares Eveline Queiroz de Pinho, Rubini Marciano Regis, Mello-de-Sousa Thiago Machado, Duarte Gilvan Caetano, de Faria Fabrícia Paula, Ferreira Filho Edivaldo Ximenes, Kyaw Cynthia Maria, Silva-Pereira Ildinete, Poças-Fonseca Marcio Jose
Department of Cellular Biology, Institute of Biological Sciences, University of Brasilia, 70.910-900 Brasilia, DF, Brazil.
Enzyme Res. 2013;2013:287343. doi: 10.1155/2013/287343. Epub 2013 Jul 10.
Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at 50°C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55°C for 72 h. Purified rEG A kinetic parameters towards CMC were determined as K m = 27.5 ± 4.33 mg/mL, V max = 1.185 ± 0.11 mmol/min, and 55.8 IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol.
构巢曲霉作为用于木质纤维素残渣降解的酶源，在生物技术领域未得到充分利用。本研究描述了构巢曲霉内切葡聚糖酶A在毕赤酵母中的异源表达、重组酶的纯化及生化特性。活性重组内切葡聚糖酶A（rEG A）作为一种35 kDa的蛋白被高效分泌，并通过两步层析法进行纯化。在50°C/pH 4条件下检测到最高酶活性。当在45°C和55°C孵育72小时时，rEG A保留了100%的活性。测定纯化后的rEG A对羧甲基纤维素（CMC）的动力学参数为：K m = 27.5 ± 4.33 mg/mL，V max = 1.185 ± 0.11 mmol/min，比活性为55.8国际单位/毫克。重组毕赤酵母上清液对香蕉茎、甘蔗渣、大豆残渣和玉米秸秆等木质纤维素残渣具有水解活性。这些数据表明，rEG A适用于将植物生物质转化为具有商业重要性的产品，如第二代燃料乙醇。
