Mutations from a family-shuffling-library reveal amino acid residues responsible for the thermostability of endoglucanase CelA from Clostridium thermocellum.
We constructed a library of chimeras from the major endoglucanase, CelA, of Clostridium thermocellum and a less stable endoglucanase CelB from Clostridium josui with multiple point mutations using low-fidelity family-shuffling method. Mutations that inactivated the enzyme were rapidly eliminated with high-throughput screening. The activities and thermostabilities of selected variants were evaluated, and four amino acid substitutions, K249R, P258S, S329N and E355G, were identified as having significant impact on the thermostability of CelA without affecting enzymatic activity. In the crystal structure of CelA, most of them are away from the activity cleft and are responsible for the stabilization of secondary structures.
Yi
Zhuo-Lin
ZL
Chengdu Institute of Biology, Chinese Academy of Sciences, P.O. Box 416, Chengdu, 610041, China.
Wu
Zhong-Liu
ZL
D019943:Amino Acid Substitution / Q000235:genetics*; D000596:Amino Acids / Q000235:genetics*; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics; D032621:DNA Shuffling; D004795:Enzyme Stability; D008958:Models, Molecular; D017354:Point Mutation; D055550:Protein Stability; D017434:Protein Structure, Tertiary
D000596:Amino Acids; C074775:CelA endoglucanase; D002480:Cellulase