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

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 20ï¼ 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.