Improved thermostability of clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis
Anbar, Michael and Gül, Özgür and Lamed, Raphael and Sezerman, Uğur and Bayer, Edward A. (2012) Improved thermostability of clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis. Applied and Environmental Microbiology, 78 (9). pp. 3458-3464. ISSN 0099-2240 (print) ; 1098-5336 (online)
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Official URL: http://dx.doi.org/10.1128/AEM.07985-11
The use of thermostable cellulases is advantageous for the breakdown of lignocellulosic biomass toward the commercial production of biofuels. Previously, we have demonstrated the engineering of an enhanced thermostable family 8 cellulosomal endoglucanase (EC 18.104.22.168), Cel8A, from Clostridium thermocellum, using random error-prone PCR and a combination of three beneficial mutations, dominated by an intriguing serine-to-glycine substitution (M. Anbar, R. Lamed, E. A. Bayer, ChemCatChem 2: 997-1003, 2010). In the present study, we used a bioinformatics-based approach involving sequence alignment of homologous family 8 glycoside hydrolases to create a library of consensus mutations in which residues of the catalytic module are replaced at specific positions with the most prevalent amino acids in the family. One of the mutants (G283P) displayed a higher thermal stability than the wild-type enzyme. Introducing this mutation into the previously engineered Cel8A triple mutant resulted in an optimized enzyme, increasing the half-life of activity by 14-fold at 85 degrees C. Remarkably, no loss of catalytic activity was observed compared to that of the wild-type endoglucanase. The structural changes were simulated by molecular dynamics analysis, and specific regions were identified that contributed to the observed thermostability. Intriguingly, most of the proteins used for sequence alignment in determining the consensus residues were derived from mesophilic bacteria, with optimal temperatures well below that of C. thermocellum Cel8A.
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