title: Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.
abstract: Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.
authors.affiliation: Division of Applied Life Sciences (BK21), PMBBRC, Gyeongsang National University, Jinju, Republic of Korea.
authors.forename: Amar A
authors.initials: AA
authors.lastname: Telke
authors.forename: Ningning
authors.initials: N
authors.lastname: Zhuang
authors.forename: Sunil S
authors.initials: SS
authors.lastname: Ghatge
authors.forename: Sook-Hee
authors.initials: SH
authors.lastname: Lee
authors.forename: Asad
authors.initials: A
authors.lastname: Ali Shah
authors.forename: Haji
authors.initials: H
authors.lastname: Khan
authors.forename: Youngsoon
authors.initials: Y
authors.lastname: Um
authors.forename: Hyun-Dong
authors.initials: HD
authors.lastname: Shin
authors.forename: Young Ryun
authors.initials: YR
authors.lastname: Chung
authors.forename: Kon Ho
authors.initials: KH
authors.lastname: Lee
authors.forename: Seon-Won
authors.initials: SW
authors.lastname: Kim
mesh_terms: D001426:Bacterial Proteins; D002480:Cellulase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D009154:Mutation; D019897:Periplasm / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D002482:Cellulose; C407024:endoglucanase Cel5A; D002480:Cellulase