direct-liquid-culture-screening-for-evaluating-production

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of <em>Aspergillus Oryzae</em>

Aspergillus oryzae, a filamentous fungus, is one of the most widely used hosts for industrial applications including large-scale production of proteins. A ...

Honda Research Institute Japan Co., Ltd.

The glycoside hydrolase family 7 (GH7) member cellobiohydrolase (CBH) is a key enzyme that degrades crystalline cellulose, an important structural component of plant cell walls. As GH7 CBH is a major component in the enzyme mixture used to degrade biomass into fermentable glucose in biorefineries, enhancing its catalytic activity will significantly impact development in this field. GH7 CBH possesses a catalytic tunnel through which cellulose substrates are threaded and hydrolysed. Despite numerous studies dissecting this processive mechanism, the role of amino acid residues in the tunnel remains not fully understood. Herein, we examined the respective contributions of nine amino acid residues in the catalytic tunnel of GH7 CBH from Talaromyces cellulolyticus by substitution with alanine. As a result, N62A and K203A mutants were found to possess significantly higher cellulase activities than wild type. Molecular dynamics simulations showed that the N62 residue interacted strongly with the cellulose substrate, impeding threading, while the N62A mutant allowed cellulose to proceed more smoothly. Furthermore, the W63 residue was observed to facilitate twisting of the cellulose substrate in our simulations. This study helps elucidate cellulose threading and provides insight into biomass hydrolysis.

Shigenobu Mitsuzawa

Honda Research Institute Japan Co., Ltd.

Direct Liquid-Culture Screening for Evaluating the Production of Heterologous Proteins Using an Auxotrophic Mutant of Aspergillus Oryzae