Enhancement of cellulase activity by clones selected from the combinatorial library of the cellulose-binding domain by cell surface engineering.
To improve the cellulolytic activity of a yeast strain displaying endoglucanase IotaIota (EG II) from Trichoderma reesei, a combinatorial library of the cellulose-binding domain (CBD) of EG II was constructed by using cell surface engineering. When EG II degrades celluloses, CBD binds to cellulose, and its catalytic domain cleaves the glycosidic bonds of cellulose. CBD had a flat face, composed of five amino acids for binding. It was supposed that the three hydrophobic amino acid residues of the five amino acid residues were essential for binding to cellulose. Therefore, by improving the two remaining amino acid residues, construction of mutants with a combinatorial library of the two amino acids in CBD was carried out and binding ability and hydrolysis activity were measured. In the first screening by halo assay using the Congo Red staining method, about 200 of the 2000 colonies formed clear halos, and then five colonies with the clearest halos were finally selected. In the second screening, the binding ability of the five mutants to phosphoric acid-swollen Avicel was measured. In addition, the measurement of hydrolysis activity toward carboxymethylcellulose (CMC) using the screened mutants was carried out. As a result, the mutated EG II exhibiting higher binding ability (1.5-fold) had higher hydrolysis activity (1.3-fold) compared to the parent EG II-displaying yeast cell, demonstrating that CBD has confirmatively some effect on the cellulase activity through its binding ability of the enzyme to cellulose.
Fukuda
Takeshi
T
Fiber Amenity Engineering, Graduate School of Engineering, University of Fukui, Japan.
Ishikawa
Tomoko
T
Ogawa
Mariko
M
Shiraga
Seizaburo
S
Kato
Michiko
M
Suye
Shin-Ichiro
S
Ueda
Mitsuyoshi
M
D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology*; D002480:Cellulase / Q000737:chemistry*; D002482:Cellulose / Q000737:chemistry; D003224:Congo Red; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D013499:Surface Properties; D014242:Trichoderma / Q000201:enzymology
D019151:Peptide Library; D003224:Congo Red; D002482:Cellulose; D002480:Cellulase