Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo Av. Trabalhador Saocarlense 400, Sao Carlos 13560-970, SP, Brazil.
J Microbiol Biotechnol. 2011 Aug;21(8):808-17. doi: 10.4014/jmb.1010.10037.
Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha- helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-β-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.
由于其较高的纤维素酶活性，丝状真菌哈茨木霉在生物质水解应用中具有相当大的潜力。哈茨木霉纤维二糖水解酶 I（ThCBHI）是一种外切葡聚糖酶，是纤维素降解过程中的一种重要酶。在这里，我们报告了一种简单的一步离子交换色谱法，用于纯化 ThCBHI 及其初步的生物物理和生化特性。通过在浸没发酵中用微晶纤维素诱导产生的 ThCBHI 在 DEAE-Sephadex A-50 介质上进行纯化，并通过质谱法确认其身份。ThCBHI 的生化特性表明，该蛋白质的分子量为 66 kDa，等电点为 5.23。通过小角 X 射线散射（SAXS）确认，全长 ThCBHI 及其用木瓜蛋白酶消化获得的催化核心结构域（CCD）在溶液中均为单体。通过圆二色性分析对 ThCBHI 的二级结构进行分析，发现α-螺旋和β-折叠的含量分别在 28%和 38%范围内。337 nm 的固有荧光发射最大值归因于 ThCBHI 色氨酸残基不同程度地暴露于水中。此外，ThCBHI 在 pH 5.0 和 50 摄氏度下显示出最大活性，对 Avicel 和对硝基苯基-β-D-纤维二糖苷的比活性分别为 1.25 U/mg 和 1.53 U/mg。
