title: Expression and functional analysis of a glycoside hydrolase family 45 endoglucanase from Rhizopus stolonifer.
abstract: A novel endoglucanase gene was cloned from Rhizopus stolonifer and expressed in Escherichia coli, the gene product EG II (45 kDa) was assigned to Glycoside Hydrolase Family 45 (GH45), and its specific activity on phosphoric acid-swollen cellulose (PASC) was 48 IU/mg. To solve the problem of substrate accumulation in the cellulose hydrolysis and enhance the catalytic efficiency of endoglucanase, the eg2 gene was modified by site directed mutagenesis. Mutations generated by overlapping PCR have been proven to increase its catalytic activity on carboxymenthyl cellulose, microcrystalline cellulose (Avicel) and PASC, among which the mutant EG II-E containing all 6 mutations (N39S, V136D, T251G, D255G, P256S and E260D) peaked 121 IU/mg on PASC. The bioinformatic analysis showed that 2 key catalytic residues (D136 and D260) moved closer with the opening of a loop after mutagenesis, and a tunnel was formed by structural transformation. This structure was conducive for the substrate to access the active centre, and D136 played an indispensable role in the substrate recognition.
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: Yingying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yaping
authors.initials: Y
authors.lastname: Yang
authors.forename: Zhewei
authors.initials: Z
authors.lastname: Song
authors.forename: Xianglin
authors.initials: X
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D019295:Computational Biology; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D002482:Cellulose; D002480:Cellulase