title: Modulating the pH-activity profile of cellulase A from Cellulomonas fimi by replacement of surface residues.
abstract: One industrial process for the production of cellulosic ethanol and or value-added products involves exposing the cellulose content of plant materials by steam explosion in the presence of strong acid, followed by its neutralization and subsequent digestion with a cocktail of cellulolytic enzymes. These enzymes typically have activity optima at slightly acidic or neutral pH and so generating enzymes that are more active and tolerant in more acidic conditions would help to reduce associated costs. Here, we describe the engineering of cellulase A from Cellulomonas fimi as a model to replace residues that were identified as potentially influencing the pH-activity profile of the enzyme based on sequence alignments and analysis of the known three-dimensional structures of other CAZy family 6 glycoside hydrolases with the aim to lower its pH optimum. Twelve specific residues and a sequence of eight were identified and a total of 30 mutant enzymes were generated. In addition to being replaced with natural amino acids, some of the identified residues were substituted with cysteine and subsequently oxidized to cysteinesulfinate. Of the four single amino acid replacements that produced enhancements of activity at acidic pH, three involved the removal of charged groups from the surface of the enzyme. The generation of double mutations provided mixed results but the combination of Glu407 → Ala and Tyr321 → Phe replacements had an additive effect on the enhancement, reaching a total activity that was 162% of the wild-type level. This study thus illustrated the utility of altering the surface charge properties of the family 6 glycoside hydrolases to enhance activity at low pH and thereby an avenue for further protein engineering.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
authors.forename: Darrell W
authors.initials: DW
authors.lastname: Cockburn
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Clarke
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D040143:Cellulomonas / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment
chemical_list: D006026:Glycoside Hydrolases; D002480:Cellulase