A thermostable and highly active fungal GH3 β-glucosidase generated by random and saturation mutagenesis.

Abstract

Enhancing the thermostability of cellulose-degrading enzymes is pivotal for establishing an efficient bioconversion system from cellulosic materials to value-added compounds. Here, by introducing random and saturation mutagenesis into the Thermoascus aurantiacus β-glucosidase gene, we generated a hyperthermostable mutant with five amino acid substitutions. Analysis of temperature-induced unfolding revealed the involvement of each replacement in the increased T_m value. Structural analysis showed that all replacements are located at the periphery of the catalytic pocket. D433N replacement, which had a pronounced thermostabilizing effect (ΔT_m ＝ 4.5°C), introduced an additional hydrogen bond with a backbone carbonyl oxygen in a long loop structure. The mutant enzyme expressed in Kluyveromyces marxianus exhibited a T_m of 82°C and hydrolyzed cellobiose with k_cat and K_m values of 200 s^－1 and 1.8 mM, respectively. When combined with a thermostable endoglucanase, the mutant enzyme released 20％ more glucose than wild-type enzyme from cellulosic material. The mutant enzyme is therefore a noteworthy addition to the existing repertoire of thermostable β-glucosidases.