Enhancement of the thermostability and activity of mesophilic Clostridium cellulovorans EngD by in vitro DNA recombination with Clostridium thermocellum CelE.
The thermal stability and catalytic activity of endoglucanase (EngD) from mesophilic Clostridium cellulovorans were improved by evolutionary molecular engineering. Thermostable mutants were isolated after staggered extension process (StEP) with celE from thermophilic Clostridium thermocellum performed to conduct family shuffling and overlay screening of the resultant mutant library. The relative activity of the best-evolved clone has been improved of about 2 times higher at 50 degrees C and showed a higher k(cat)/K(m) value than its engD parental clone. We determined that these variants had two amino acid substitutions (L157N, Q158E) and confirmed their effects by substituting these amino acids in the parental gene by site-directed mutagenesis. These substitutions resulted in an increase in hydrophilic or charged residues. Our results demonstrate that in vitro recombination is an effective approach to improve the thermostability and enzymatic activity of a mesophilic enzyme.
Lee
Chae Yoeng
CY
School of Life Science and Biotechnology, Korea University, Seoul 136-713, Republic of Korea.
Yu
Kyung Ok
KO
Kim
Seung Wook
SW
Han
Sung Ok
SO
D019943:Amino Acid Substitution; D001483:Base Sequence; D044602:Cellulases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D046974:Clostridium cellulovorans / Q000201:enzymology* / Q000235:genetics*; D048013:Acetivibrio thermocellus / Q000201:enzymology* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D044602:Cellulases