Improved Thermostability of Clostridium thermocellum Endoglucanase Cel8A by Using Consensus-Guided Mutagenesis
Open Access
- 1 May 2012
- journal article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 78 (9), 3458-3464
- https://doi.org/10.1128/aem.07985-11
Abstract
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 3.2.1.4), 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°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.Keywords
This publication has 39 references indexed in Scilit:
- Applications of computational science for understanding enzymatic deconstruction of celluloseCurrent Opinion in Biotechnology, 2011
- Design of Thermostable Beta-Propeller Phytases with Activity over a Broad Range of pHs and Their Overproduction by Pichia pastorisApplied and Environmental Microbiology, 2010
- The Carbohydrate-Active EnZymes database (CAZy): an expert resource for GlycogenomicsNucleic Acids Research, 2008
- Structure‐guided consensus approach to create a more thermostable penicillin G acylaseBiotechnology Journal, 2006
- Scalable molecular dynamics with NAMDJournal of Computational Chemistry, 2005
- Extending the treatment of backbone energetics in protein force fields: Limitations of gas‐phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulationsJournal of Computational Chemistry, 2004
- The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 å resolution: structural characterization of proline-substitution sites for protein thermostabilizationJournal of Molecular Biology, 1997
- VMD: Visual molecular dynamicsJournal of Molecular Graphics, 1996
- Protein Unfolding Pathways Explored Through Molecular Dynamics SimulationsJournal of Molecular Biology, 1993
- Use of Dinitrosalicylic Acid Reagent for Determination of Reducing SugarAnalytical Chemistry, 1959