Tang Bin, Wei Lanfang, Tang Wenjing, Li Song, Zhou Ruofei
College of Biochemical Engineering, Anhui Polytechnic University, Wuhu, 241000, China,
Appl Biochem Biotechnol. 2015 Aug;176(8):2242-52. doi: 10.1007/s12010-015-1711-2. Epub 2015 Jul 26.
This study investigated an endoglucanase (EGII) from Rhizopus stolonifer var. reflexus TP-02 that consists of a C-terminal catalytic domain and an N-terminal carbohydrate-binding module joined by a linker rich in glycine, serine, threonine, and alanine. Site-directed mutagenesis was applied to characterize the conformation and dynamics of the linker. Mutants were expressed in Escherichia coli BL21 and purified by Ni-chelating column. Structural analysis indicated that glycine provided flexibility in the enzymatic process. G67P, G91Y, G101Y, G108Y, G109Y, G112P, H61G, H75G, and Y103G were selected on the basis of the results of the bioinformatics and Ramachandran plot analysis for the linker. The catalytic activities of EGII and its mutants on CMC-Na, microcrystalline cellulose (Avicel), and phosphoric acid-swollen celluloses (PASC) showed that flexible amino acids strengthened the activity of the enzyme. It indicated that flexible amino acids could improve the flexibility of the linker. Overall, the linker affected the catalytic efficiency of the endoglucanase in hydrolyzing cellulose chains.
本研究对来自匍枝根霉变种reflexus TP - 02的一种内切葡聚糖酶（EGII）进行了研究，该酶由一个C端催化结构域和一个N端碳水化合物结合模块组成，二者通过富含甘氨酸、丝氨酸、苏氨酸和丙氨酸的连接子相连。应用定点诱变来表征连接子的构象和动力学。突变体在大肠杆菌BL21中表达，并通过镍螯合柱进行纯化。结构分析表明，甘氨酸在酶促过程中提供了灵活性。根据生物信息学结果和连接子的拉氏构象图分析，选择了G67P、G91Y、G101Y、G108Y、G109Y、G112P、H61G、H75G和Y103G。EGII及其突变体对羧甲基纤维素钠（CMC - Na）、微晶纤维素（微晶纤维素）和磷酸膨胀纤维素（PASC）的催化活性表明，柔性氨基酸增强了酶的活性。这表明柔性氨基酸可以提高连接子的灵活性。总体而言，连接子影响内切葡聚糖酶水解纤维素链的催化效率。
