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

Abstract

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.

Psychrophilic lifestyles: mechanisms of adaptation and biotechnological tools

“…For a recent in-depth review see Barroca et al (2017a). Some of the better known commercial successes include a cold-adapted lipase used in the organic synthesis of various pharmaceutical, cosmetic and flavor compounds (Kirk and Christensen 2002); a xylanase for enhancing bread quality (Barroca et al 2017b;Collins et al 2012;Collins et al 2006;Collins et al 2002b;Dutron et al 2010Dutron et al -2012; various hydrolases in detergents for low temperature cleaning (Sarmiento et al 2015), and various enzymes (alkaline phosphate, nuclease and uracil-DNA N-glycosylase) used in molecular biology for their high activity and ease of inactivation (Barroca et al 2017a).…”

Candida albicans chitinase 3 with potential as a vaccine antigen: production, purification, and characterisation

“…The temperature dependence of activity was analysed between 10 and 70°C with a 5 min activity assay. The pH dependence of activity was determined between pH 1.5 and 12.5 with buffer mix (100 mM Citrate (Merck), 20 mM MES (Sigma), 20 mM MOPS (Sigma), 20 mM TAPS (Sigma), 20 mM CHES (Sigma), and 20 mM CAPS (Sigma) [ 16 ] and a 5 min activity assay.…”

“…Fluorescence intensity of 30 µg mL -1 Cht3 was measured at 345 nm with excitation at 280 nm during a temperature ramp from 25 • C to 90 • C at 1 • C/min, and data were treated as described by Pace. [17] Reversibility in the loss of tertiary structure was assessed by com-paring fluorescence emission spectra (300 to 400 nm, excitation at 280 nm) of undenatured protein with that of protein that had been heated at 1 • C/min to the denaturation temperature (Tm), or to 90 • C, before being re-cooled at 1 • C/min to 25 • C. To analyse the structural stability as a function of pH, Cht3 (300 µg mL -1 ) was adjusted to pHs from 2 to 13 by 10-fold dilution in buffer mix, [16] and the 300 to 400 nm fluorescence emission spectra with excitation at 280 nm measured following 1 and 7 days incubation at 18 • C. The fluorescence emission spectra of the following were also measured as controls: 50 mM HEPES buffer, pH 7.5, diluted 10-fold in buffer mix at pH 7.5, and Cht3 (30 µg mL -1 ) heated at 1 • C/min to 90 • C.…”