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 degreesC) by a margin of 73 degreesC as compared with the native enzyme (T-opt = 49 degreesC) 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 degreesC, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55 degreesC, 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.