Site-directed mutagenesis and CBM engineering of Cel5A (Thermotoga maritima).
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.
Mahadevan
Shobana Arumugam
SA
Department of Forest Products and Technology, BK21 Program, Chonnam National University, Kwangju, Korea.
Wi
Seung Gon
SG
Lee
Dae-Seok
DS
Bae
Hyeun-Jong
HJ