Deciphering the factors defining the pH-dependence of a commercial glycoside hydrolase family 8 enzyme.

A prerequisite to the use of any enzyme in any industrial process is an understanding of its activity and stability under process conditions. Glycoside hydrolase family 8 enzymes include many important biotechnological biocatalysts yet little is known of the performance of these with respect to pH. A better understanding of this parameter and its relationship to structure and function in these enzymes will allow for an improved use of these in industry as well as an enhanced ability in their engineering and optimisation for a particular application. An in-depth analysis of the pH induced changes in activity, irreversible inactivation, conformation, stability and solubility of a commercial glycoside hydrolase family 8 xylanase was carried out with the aim of identifying the factors determining the pH dependence of this enzyme. Our study showed that different phenomena play different roles at the various pHs examined. Both reversible and irreversible processes are involved at acidic pHs, with the irreversible processes dominating and being due to protein aggregation and precipitation. At basic pHs, loss of activity is principally due to reversible processes, possibly deprotonation of an essential catalytic residue, but at higher pHs, near the pI of the protein, precipitation again dominates while structure unfolding was discerned at the higher pHs investigated. Such insights demonstrate the complexity of factors involved in the pH dependence of proteins and advances our knowledge on design principles and concepts for engineering proteins. Our results highlight the major role of protein precipitation in activity and stability losses at both low and high pHs but it is proposed that different strategies be used in tailoring the enzyme to overcome this in each case. Indeed the detailed understanding obtained here will allow for a more focused, informed and hence successful tailoring of glycoside hydrolase family 8 proteins for a specific pH and process application.