Departamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade de São Paulo, Lorena, SP, Brazil.
Laboratório Nacional de Ciência e Tecnologia do Bioetanol, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP, Brazil.
Enzyme Microb Technol. 2019 Jan;120:23-35. doi: 10.1016/j.enzmictec.2018.09.005. Epub 2018 Sep 27.
Lignocellulosic materials are abundant, renewable and are emerging as valuable substrates for many industrial applications such as the production of second-generation biofuels, green chemicals and pharmaceuticals. However, the recalcitrance and the complexity of cell wall polysaccharides require multiple enzymes for their complete conversion to oligo- and monosaccharides. The endoglucanases from GH45 family are a small and relatively poorly studied group of enzymes with potential industrial application. The present study reports cloning, heterologous expression and functional characterization of two GH45 endoglucanases from mesophilic fungi Gloeophyllum trabeum (GtGH45) and thermophilic fungi Myceliophthora thermophila (MtGH45), which belong to subfamilies GH45C and GH45A, respectively. Both enzymes have optimal pH 5.0 and melting temperatures (Tm) of 66.0 °C and 80.9 °C, respectively, as estimated from circular dichroism experiments. The recombinant proteins also exhibited different mode of action when incubated with oligosaccharides ranging from cellotriose to cellohexaose, generating mainly cellobiose and cellotriose (MtGH45) or glucose and cellobiose (GtGH45). The MtGH45 did not show activity against oligosaccharides smaller than cellopentaose while the enzyme GtGH45 was able to depolymerize cellotriose, however with lower efficiency when compared to larger oligosaccharides. Furthermore, both GHs45 were stable up to 70 °C for 24 h and useful to enhance initial glucan hydrolysis rates during saccharification of sugarcane pith by a mixture of cellulolytic enzymes. Recombinant GHs45 from diverging subfamilies stand out for differences in substrate specificity appearing as new tools for preparation of enzyme cocktails used in cellulose hydrolysis.
木质纤维素材料丰富、可再生，并且正在成为许多工业应用（如第二代生物燃料、绿色化学品和制药）的有价值的基质。然而，细胞壁多糖的顽固性和复杂性需要多种酶才能将其完全转化为寡糖和单糖。GH45 家族的内切葡聚糖酶是一组小而研究相对较少的酶，具有潜在的工业应用前景。本研究报告了嗜热真菌 Myceliophthora thermophila（MtGH45）和中温真菌 Gloeophyllum trabeum（GtGH45）中两种 GH45 内切葡聚糖酶的克隆、异源表达和功能特性。这两种酶的最适 pH 值均为 5.0，根据圆二色性实验估计的熔点（Tm）分别为 66.0°C 和 80.9°C。重组蛋白在与从纤维三糖到纤维六糖的寡糖孵育时也表现出不同的作用模式，主要产生纤维二糖和纤维三糖（MtGH45）或葡萄糖和纤维二糖（GtGH45）。MtGH45 对小于纤维五糖的寡糖没有活性，而酶 GtGH45 能够降解纤维三糖，但其效率低于较大的寡糖。此外，两种 GH45 在 70°C 下稳定 24 小时，并且在甘蔗髓的纤维素酶糖化过程中，能够提高初始葡聚糖水解率，这两种 GH45 都很有用。来自不同亚家族的重组 GH45 因其底物特异性的差异而引人注目，它们似乎是用于纤维素水解的酶混合物制备的新工具。
