Voutilainen Sanni P, Puranen Terhi, Siika-Aho Matti, Lappalainen Arja, Alapuranen Marika, Kallio Jarno, Hooman Satu, Viikari Liisa, Vehmaanperä Jari, Koivula Anu
VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Finland.
Biotechnol Bioeng. 2008 Oct 15;101(3):515-28. doi: 10.1002/bit.21940.
As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-10 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70 degrees C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes.
作为为生物乙醇生产工艺寻找更好的纤维素酶工作的一部分，我们一直在寻找新型的GH-7家族纤维二糖水解酶，这类酶对不溶性聚合物底物具有特别高的活性，并参与纤维素水解的限速步骤。本文研究并报道了嗜热真菌嗜热顶孢霉、橙黄嗜热子囊菌和嗜热毛壳菌中7家族纤维二糖水解酶的酶学性质。实验中以里氏木霉Cel7A酶作为参照。由于天然的橙黄嗜热子囊菌Cel7A没有碳水化合物结合模块（CBM），因此还构建了含有来自嗜热毛壳菌Cel7A或里氏木霉Cel7A的CBM的重组蛋白。所有这些新型酸性纤维二糖水解酶在45℃下对微晶纤维素（微晶纤维素）的水解比里氏木霉Cel7A更耐热（高4-10摄氏度）且活性更高（两到四倍）。当以可溶性底物进行测定时，嗜热毛壳菌Cel7A表现出最高的比活性和最适温度。然而，在70℃下对微晶纤维素水解最有效的酶是橙黄嗜热子囊菌Cel7A的双模块版本，它也相对较少受到纤维二糖的抑制。基于这些酶的三维同源模型，从结构角度对这些结果进行了讨论。
