Szijártó Nóra, Siika-Aho Matti, Tenkanen Maija, Alapuranen Marika, Vehmaanperä Jari, Réczey Kati, Viikari Liisa
Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, P.O. Box 91, H-1521 Budapest, Hungary.
J Biotechnol. 2008 Sep 10;136(3-4):140-7. doi: 10.1016/j.jbiotec.2008.05.010. Epub 2008 May 28.
Three thermostable neutral cellulases from Melanocarpus albomyces, a 20-kDa endoglucanase (Cel45A), a 50-kDa endoglucanase (Cel7A), and a 50-kDa cellobiohydrolase (Cel7B) heterologously produced in a recombinant Trichoderma reesei were purified and studied in hydrolysis (50 degrees C, pH 6.0) of crystalline and amorphous cellulose. To improve their efficiency, M. albomyces cellulases naturally harboring no cellulose-binding module (CBM) were genetically modified to carry the CBM of T. reesei CBHI/Cel7A, and were studied under similar experimental conditions. Hydrolysis performance and product profiles were used to evaluate hydrolytic features of the investigated enzymes. Each cellulase proved to be active against the tested substrates; the cellobiohydrolase Cel7B had greater activity than the endoglucanases Cel45A and Cel7A against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence of synergism was observed when mixtures of the novel enzymes were applied in a constant total protein dosage. Presence of the CBM improved the hydrolytic potential of each enzyme in all experimental configurations; it had a greater effect on the endoglucanases Cel45A and Cel7A than the cellobiohydrolase Cel7B, especially against crystalline substrate. The novel cellobiohydrolase performed comparably to the major cellobiohydrolase of T. reesei (CBHI/Cel7A) under the applied experimental conditions.
从重组里氏木霉中异源产生的来自白黑侧耳的三种热稳定中性纤维素酶，一种20 kDa的内切葡聚糖酶（Cel45A）、一种50 kDa的内切葡聚糖酶（Cel7A）和一种50 kDa的纤维二糖水解酶（Cel7B），被纯化并用于研究其在50℃、pH 6.0条件下对结晶纤维素和无定形纤维素的水解作用。为了提高它们的效率，对白黑侧耳中天然不含纤维素结合模块（CBM）的纤维素酶进行基因改造，使其携带里氏木霉CBHI/Cel7A的CBM，并在相似的实验条件下进行研究。水解性能和产物谱用于评估所研究酶的水解特性。每种纤维素酶对测试底物均表现出活性；纤维二糖水解酶Cel7B对结晶纤维素的活性高于内切葡聚糖酶Cel45A和Cel7A，而对于无定形底物，顺序则相反。当以恒定的总蛋白剂量应用新型酶的混合物时，观察到协同作用的证据。在所有实验配置中，CBM的存在均提高了每种酶的水解潜力；它对内切葡聚糖酶Cel45A和Cel7A的影响比对纤维二糖水解酶Cel7B的影响更大，尤其是对结晶底物。在所应用的实验条件下，新型纤维二糖水解酶的表现与里氏木霉的主要纤维二糖水解酶（CBHI/Cel7A）相当。
