Assessing LPMO Activity on Chitin and Cellulose

LPMO activity was primarily assessed for β-chitin (10 g/L), using 1 µM enzyme and 1 mM ascorbic acid in 50 mM sodium phosphate buffer, pH 6.0. Unless stated otherwise, all reactions were incubated in 2 mL Eppendorf tubes at 800 rpm in an Eppendorf thermomixer set to 40 °C (Eppendorf, Hamburg, Germany). Extracted and deproteinized β-chitin from squid pen (batch 20,140,101, France Chitin, Orange, France), was ball-milled and sieved to produce separate fractions of particles with 75–200 μm and 500–850 μm size. Activity was also tested towards 10 g/L shrimp shell (Pandalus borealis) α-chitin [purchased from Chitinor AS; Senjahopen, Norway; demineralized by hydrochloric acid treatment and subsequently deproteinized by alkaline (NaOH) treatment] and cellulosic substrates such as 5 g/L phosphoric acid swollen cellulose (PASC; prepared from Avicel PH-101 as described by Wood^{83 (link)}); 10 g/L Avicel PH-101 (Sigma-Aldrich, St. Louis, MO, USA) and 1 g/L bacterial microcrystalline cellulose^{84 (link)} (kindly provided by Dr. Priit Väljamäe, University of Tartu). Furthermore, activity was tested towards chitin oligomers (DP5-6, 2 mM) purchased from Megazyme (Bray, Ireland), and peptidoglycan isolated from Streptomyces sp. (3 g/L) purchased from Merck (Darmstadt, Germany).