Coupling of Surface Carboxyls of Carboxymethylcellulase with Aniline via Chemical Modification: Extreme Thermostabilization in Aqueous and Water-Miscible Organic Mixtures

We wish to report the attainment of the highest ever Topt by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in Vmax» Km, and optimum pH! This extraordinary enhancement in thermophilicity of anilinecoupled CMCase (Topt = 122 °C) by a margin of 73 °C as compared with the native enzyme (Topt = 49 °C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis. It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents. The Topt of native CMCase and ACC were 25 and 90 °C, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55 °C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min. We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic—aromatic interactions and