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 T_opt by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in V_max, K_m, and optimum pH! This extraordinary enhancement in thermophilicity of aniline-coupled CMCase (T_opt = 122°C) by a margin of 73°C as compared with the native enzyme (T_opt = 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 T_opt 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 aromatic interactions with the sugar backbone of the substrate, respectively.