title: Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima).
abstract: In order to make cost-effective bioethanol from dynamic lignocellulosic material, we require potentially acting and stable cellulolytic enzymes. In our investigation, the hyperthermostable endoglucanase Cel5A from Thermotoga maritima was subjected to site-directed mutagenesis and carbohydrate-binding module (CBM) engineering. For this purpose, amino acids around the active-site region were targeted. Results indicated that five single mutants showed a shift in optimal pH from 5 to 5.4. The N147E mutant displayed 10% higher activity than native Cel5A. Domain engineering was performed with fungal and bacterial CBM. In addition, CBM1 from (CBHII) Trichoderma reesei and CBM6 from Clostridium stercorarium xylanase A were fused with Cel5A. Both the CBM-engineered Cel5A showed 14-18-fold higher hydrolytic activity towards Avicel. Immuno-gold labeling assay of engineered enzymes further indicated the relativity that exists between binding ability and activity.
authors.affiliation: Department of Forest Products and Technology, BK21 Program, Chonnam National University, Kwangju, Korea.
authors.forename: Shobana Arumugam
authors.initials: SA
authors.lastname: Mahadevan
authors.forename: Seung Gon
authors.initials: SG
authors.lastname: Wi
authors.forename: Dae-Seok
authors.initials: DS
authors.lastname: Lee
authors.forename: Hyeun-Jong
authors.initials: HJ
authors.lastname: Bae