Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
PLoS One. 2012;7(11):e50488. doi: 10.1371/journal.pone.0050488. Epub 2012 Nov 30.
Glucoamylases, containing starch-binding domains (SBD), have a wide range of scientific and industrial applications. Random mutagenesis and DNA shuffling of the gene encoding a starch-binding domain have resulted in only minor improvements in the affinities of the corresponding protein to their ligands, whereas circular permutation of the RoCBM21 substantially improved its binding affinity and selectivity towards longer-chain carbohydrates. For the study reported herein, we used a standard soluble ligand (amylose EX-I) to characterize the functional and structural aspects of circularly permuted RoCBM21 (CP90). Site-directed mutagenesis and the analysis of crystal structure reveal the dimerisation and an altered binding path, which may be responsible for improved affinity and altered selectivity of this newly created starch-binding domain. The functional and structural characterization of CP90 suggests that it has significant potential in industrial applications.
葡糖淀粉酶含有淀粉结合结构域（SBD），具有广泛的科学和工业应用。对编码淀粉结合结构域的基因进行随机诱变和 DNA 改组，仅能使相应蛋白质与配体的亲和力略有提高，而 RoCBM21 的环状排列则大大提高了其对长链碳水化合物的结合亲和力和选择性。在本文报道的研究中，我们使用标准的可溶性配体（支链淀粉 EX-I）来表征环状排列的 RoCBM21（CP90）的功能和结构方面。定点突变和晶体结构分析揭示了二聚化和改变的结合途径，这可能是导致这个新创建的淀粉结合结构域提高亲和力和改变选择性的原因。CP90 的功能和结构表征表明，它在工业应用中具有重要的潜力。
