title: Effect of Linker Flexibility on the Catalytic Features of a Glycoside Hydrolase Family 45 Endoglucanase from Rhizopus stolonifer.
abstract: 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.
authors.affiliation: College of Biochemical Engineering, Anhui Polytechnic University, Wuhu, 241000, China, tangbin@ahpu.edu.cn.
authors.forename: Bin
authors.initials: B
authors.lastname: Tang
authors.forename: Lanfang
authors.initials: L
authors.lastname: Wei
authors.forename: Wenjing
authors.initials: W
authors.lastname: Tang
authors.forename: Song
authors.initials: S
authors.lastname: Li
authors.forename: Ruofei
authors.initials: R
authors.lastname: Zhou
mesh_terms: D000327:Adsorption; D000595:Amino Acid Sequence; D055162:Biocatalysis; D019295:Computational Biology; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology*; D020539:Sequence Analysis, Protein; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases