Ahmed S, Luís A S, Brás J L A, Fontes C M G A, Goyal A
Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India.
Biochemistry (Mosc). 2013 Nov;78(11):1272-9. doi: 10.1134/S0006297913110072.
The gene encoding the family 6 carbohydrate-binding module (CtCBM6A) from Clostridium thermocellum, cloned in pET-21a(+) expression vector, was overexpressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant CtCBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of CtCBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (Ka of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (Ka of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However, CtCBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant Ka of 230 M(-1) and binding capacity (N0) of 11 µmole/g. The protein melting curve of CtCBM6A displayed a peak shift from 53 to 58°C in the presence of Ca2+, indicating that Ca2+ imparts thermal stability to the CtCBM6A structure. Homology modeling of CtCBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of CtCBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that CtCBM6A has a stable conformation.
编码来自嗜热栖热菌（Clostridium thermocellum）的6型碳水化合物结合模块（CtCBM6A）的基因，克隆于pET-21a(+)表达载体中，利用大肠杆菌BL-21(DE3)细胞进行了过量表达，并通过固定化金属离子亲和层析进行了纯化。重组CtCBM6A的SDS-PAGE分析显示其分子大小约为15 kDa。通过亲和凝胶电泳对CtCBM6A与黑麦阿拉伯木聚糖和燕麦speltoid木聚糖进行配体结合分析，结果显示其对这些配体的亲和力较低（Ka分别为40和26升/克），荧光光谱分析（Ka分别为33和15升/克）也证实了其与上述可溶性配体的结合亲和力较低。然而，CtCBM6A与不溶性小麦阿拉伯木聚糖显示出显著更高的配体结合亲和力，其平衡缔合常数Ka为230 M(-1)，结合容量（N0）为11微摩尔/克。在存在Ca2+的情况下，CtCBM6A的蛋白质熔解曲线显示峰值从53°C移至58°C，表明Ca2+赋予了CtCBM6A结构热稳定性。CtCBM6A的同源建模揭示了一种特征性的β-三明治核心结构。CtCBM6A的拉氏图显示89%的残基位于最有利区域，10%位于额外有利区域，1%位于宽松允许区域，表明CtCBM6A具有稳定的构象。
