Modulation of the pH-activity profile of the endoglucanase CenA from Cellulomonas fimi through rational design and chemical modification

Cellulose is the most abundant biopolymer on Earth and as such its efficient utilization is a key part of any sustainable approach to resource management. However, it is a highly recalcitrant molecule that is most efficiently degraded through the use of biocatalysts. Thus it is important to understand the structure-function relationships within the enzymes responsible for this degradation so they can be tailored for specific applications. One of the key properties of these enzymes is the pH dependence of their activity. This study has sought to elucidate the determinants of the pH profile for the endoglucanase CenA from Cellulomonas fimi.;A model of CenA and an alignment of the protein with related enzymes were also used to identify residues that might be important for controlling the pH profile of this enzyme. Substitutions were conducted at these positions and four were identified that were significantly more active than the wild-type enzyme at acidic pH values. These substitutions were Lys292Ala, Tyr321Phe, Glu363Ala and Glu399Ala. Combinations of these substitutions were constructed, which produced enzymes with up to 160% of the activity of the wild-type enzyme at pH 5. Finally, random mutagenesis was used to identify other positions within the enzyme that are important for determining the pH profile. Using this method, four substitutions were identified that were more active than the wild-type enzyme at pH 5. Taken together these results have provided an important contribution to the development of rational engineering approaches for cellulolytic enzymes.;One approach that was utilized was the replacement of the catalytic general base of CenA (Asp392) with cysteinesulfinate. This amino acid is similar in structure to aspartate, but has a lower pKa, thus improving its ability to act as a general base at low pH values. The result was an increase in activity at low pH values, exceeding wild-type activity below pH 5.5, reaching 300% of wild-type activity at pH 4.5. This also provided confirmation of the identity of the catalytic base in this enzyme. This approach was extended to other endoglucanases as well, in order to demonstrate the general applicability of replacing the catalytic base with cysteinesulfinate.