Avicel ph 101

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).

Avicel^® PH-101 (Sigma), microcrystalline cellulose and dissolving pulp (DP, pure cellulosic substrate less than 0.5 % lignin, less than 3 % xylan) [35 (link)] from Canadian Ponderosa Pine were used as substrate for cellulolytic hydrolysis and SSF by the cellobiose-utilizing C. glutamicum strains. Each cellulolytic hydrolysis and SSF was carried out in CgXII medium (pH 7.0) with 1 % (w/v) Avicel^® PH-101 or 1 % (w/v) dissolvping pulp at 30 °C and 200 rpm. Celluclast 1.5 L (Sigma; Cat C2730) [75 filter paper unit (FPU)/g-glucan] was used as the cellulolytic enzymes for saccharification of Avicel^® PH-101 or DP. Cellulase actitivity of the Celluclast 1.5 L was determined by the standard filter paper assay with the 3,5-dinitrosalicylic acid (DNS) method [36 ]. One unit of cellulose activity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per mint at pH 4.8 and 50 °C. The enzyme activity of Celluclast 1.5 L was measured to be 28 FPU/mL. A colorimetric method based on the phenol–sulfuric acid reaction was used to determine the amount of residual substrate during SSF by quantifying total sugars [37 (link)].

Avicel PH101 (Sigma-Aldrich) was washed four times in MilliQ water and three times in 50 mM acetate buffer pH 5.0 (henceforth called standard buffer). Phosphoric acid swollen cellulose (PASC) was produced from Avicel PH101 (Sigma-Aldrich) as described previously (59 ). The PASC suspension was divided into two stocks; one was washed three times in standard buffer and resuspended in standard buffer, this stock is referred to as PASC, the other stock was treated with CuSO₄ as described below and referred to as oxidized PASC.