Rapid Screening of Additive Formulations for Enhancing Xylanase Stability in Pulp Bleaching and Storage Conditions

Abstract

Enzyme deactivation is a significant parameter to restrain the industrial commercialization of enzymes. In this study, a high-throughput screening technique using differential scanning fluorimetry (DSF) incorporated with enzyme activity analysis was thus developed in order to identify a suitable additive formulation for promoting enzyme stability in terms of conformational stability and functional activity. To demonstrate the concept feasibility, endo-xylanase obtained from metagenome of termite gut-inhabiting bacteria (XYN12) was used as a study model and several group of additives including salts, polymers, polyols and sugars at various concentrations and different combination patterns (single, double and triple pairing) were applied for investigating their effects on enzyme stability. As a result, a total of 80 reaction mixtures were retrieved including a native control (enzyme without any additives). DSF assay was performed using real-time PCR instrument to detect unfolding of protein in the presence of a florescence dye. Accurate melting temperature (T_m) values of each enzyme formulation were calculated using Boltzmann equation and compared to the T_m of the native control in order to find ?T_m. After that, the residual activity of xylanase in all reaction mixtures were measured using pHBAH method in automated liquid handling system. For single additives, glycerol had a stabilizing effect to the target enzyme, as observed by a significant increase in the ?T_m, while ?(NH₄)₂SO₄, CaCl₂, NaCl destabilized it. In addition, cocktail additives included glycerol, PEG4000 and trehalose dramatically enhanced the enzyme stability by positive shifting of T_m from 54 to 56 ⁽C and increasing of relative activity by more than 4 folds compared to single additive. Storage stability of the enzyme was improved when mixed with this cocktail additive as indicated by remaining of the enzyme activity (86 %) after kept at 30 ⁽C, pH 7.4 for 262 days, whereas native control lost almost of its activity within 40 days when stored at the same condition.