From bench to biorefinery: custom cellulolytic cocktail development for 2G ethanol

Abstract

This study reports the development of a novel and cost-effective cellulolytic enzyme cocktail, named Remzyme, using Rasamsonia emersonii. By supplementing the heterologously expressed carbohydrate-active enzymes (CAZymes) such as lytic polysaccharide monooxygenase (Rem_LPMO1, Rem_GH7CBHI), and xylanase (Malci_GH10xyl), the cocktail was optimized using a Simplex lattice mixture design. This innovative blend achieved a saccharification efficiency of 98.59% when applied to unwashed, acid/steam-pretreated rice straw slurry sourced from an industrial-scale 2G ethanol plant. The process was conducted under industrially relevant conditions with 15% substrate loading and protein loading of 8 mg/g dry substrate. Remarkably, the Remzyme cocktails was comparable to the leading commercial enzyme mix, CellicCTec3, at equivalent protein loadings, underscoring its potential as a cost-effective alternative in enzymatic saccharification. The study demonstrates the synergistic efficacy of accessory enzymes and core cellulases, offering significant advancements in enzyme technology for biorefinery applications.