Microbial Expansin Related Proteins for Improved Enzymatic Action for Conversion of Lignocellulose to Platform Sugars

Cost effective enzymatic deconstruction of cellulosic materials for the production of renewable fuels and chemicals is challenged by the limited accessibility of enzymes to cellulose substrates. Recently identified loosenin-like proteins (PcaLOOLs from Phanerochaete carnosa) can promote amorphogenesis and disrupt noncovalent binding between cellulose fibrils, thus promoting cell wall loosening and increasing surface accessibility for enzymatic hydrolysis. Like other microbial expansin-related proteins characterized to date, loosenins do not display hydrolytic activity; instead, they reportedly boost hydrolysis by cellulolytic enzymes although the impact of expansin-related proteins on cellulolytic activity is substrate dependent (Liu et al., 2015).

Herein, the impact of four PcaLOOLs on the activity of different cellulolytic enzymes was evaluated using a lignocellulosic substrate from softwood fiber. The PcaLOOLs were heterologously produced in Pichia pastoris in shake-flask and 5-L bioreactor systems, and then purified by affinity chromatography. Notably, the bioreactor production increased PcaLOOL yield over 10-times. Each PcaLOOL was used to pretreat the lignocellulosic material prior to the addition of cellulolytic enzyme; alternatively, each PcaLOOL was added directly with the cellulolytic enzyme to the lignocellulosic substrate. The release of soluble sugars was quantified at regular time points over 24-hours using the 4-hydroxybenzoic acid hydrazide (PAHBAH) assay and by high-performance anion exchange chromatography/pulsed amperometric detection (HPAEC-PAD). The hydrolysis reactions were performed at 40 °C and 50 °C to evaluate the impact of temperature on the boosting performance of PcaLOOLs. Briefly, all PcaLOOLs
increased the enzymatic release of soluble sugars from the lignocellulosic substrate. On-going HPAEC analyses will evaluate the impact of PcaLOOLs on the profile of sugars released from the lignocellulosic substrate. In addition to lignocellulose deconstruction, the potential of PcaLOOLs to promote fiber
defibrillation will be investigated.