Aromatic residues surrounding the active site tunnel of TfCel48A influence activity, processivity, and synergistic interactions with other cellulases

Abstract

Cel48A of Thermobifida fusca (TfCel48A) is a processive exocellulase that contains an active site tunnel and digests lignocellulosic biomass via synergistic interactions between different cellulases. Cel48A possesses a number of aromatic amino acids lining the tunnel entrance, which are highly conserved across a diverse number of microbial species and appear to play a role in the selection and threading of individual strands of cellulose from highly recalcitrant substrates. In this study, we sought to further elucidate the roles of these tunnel entrance aromatic amino acids by creating a series of double mutants and examining their effect on TfCel48A activity, processivity, and synergistic interactions with the well-studied processive endocellulase TfCel9A. Our results provide further insight concerning the mechanism of Cel48A kinetics with soluble and insoluble substrates and could play an influential role in the application of Cel48A and other exocellulases for industrial purposes. K E Y W O R D Sactive site tunnel, exocellulase, recalcitrant cellulose, synergism, Thermobifida fusca

Coevolutionary analysis reveals a distal amino acid residue pair affecting the catalytic activity of GH5 processive endoglucanase from Bacillus subtilis BS‐5

“…Cellulose is the most abundant and sustainable carbohydrate product on earth, and the efforts to convert it into fermentable sugars might provide renewable energy on a scale (Hefferon et al, 2019). The processivity of cellulases is a common feature of many exoglucanases (Basit & Akhtar, 2018) and is defined by its ability to remain attached to the strand and continuously repeat cycles of catalysis with a simultaneous release of the soluble oligosaccharides before dissociation (K. D. Zhang et al, 2018).…”

Engineering the Relatively Conserved Residues in Active Site Architecture of Thermophilic Chitinase SsChi18A Enhanced Catalytic Activity

“…For processive enzymes, the aromatic residues lining the active subsites formed a flexible hydrophobic sheath, providing a glide track for processive action. 12,14,33,34 The conservatism of the 13 aromatic residues differed at each subsite in the active site architecture. Of these, Tyr 1C), which had design potential.…”

Integration of synthetic microbial consortia based bioprocessing with pyrolysis for efficient conversion of cellulose to valuables

“…TfCel48A is another unique exocellulase from T. fusca, which is proposed to work in synergy with TfCel9A, creating a positive feedback mechanism. 81,82 The hybrid minimal-waste process involving high temperature pyrolysis of the spent fermentation culture, yielded a carbon material with physicochemical properties similar to commercial activated carbon. 83 This could be employed for making water filtration columns, various adsorbents and even energy storage devices.…”

“…TfCel48A is another unique exocellulase from T. fusca , which is proposed to work in synergy with TfCel9A, creating a positive feedback mechanism. 81,82…”