Variants of the Thermoascus aurantiacus Eg1 enzyme with higher catalytic efficiency than wild-type were obtained via site-directed mutagenesis. The mutations were chosen based on a rational mutagenesis strategy including extensive family analysis, modeling, and literature survey. Two promising variants were constructed and the enzymes were expressed and secreted from Pichia pastoris. The two mutants showed 1.7- and 4.0- fold increases in kcat with 1.5- and 2.5-fold improvements in hydrolytic activity on cellulosic substrates, respectively, while maintaining thermostability. Similar to the parent, the two variants were active between pH 4.0� 8.0 and showed optimal activity at temperature 70�C, pH 5.0. In contrast, no improvement in hydrolysis was observed with a third variant carrying a combination of both mutations, which instead showed a 60% reduction in catalytic efficiency. This work further demonstrates that non-catalytic amino acid residues can be engineered to enhance catalytic efficiency in pretreatment enzymes of interest.

Novel mutants of the <i>Thermoascus aurantiacus </i>endo-β-1,4 glucanase Eg1 with increased specific activity by rational mutagenesis

243rd National Meeting (2012)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.

Novel mutants of the Thermoascus aurantiacus endo-β-1,4 glucanase Eg1 with increased specific activity by rational mutagenesis