Laminaripentaose

Lichenan from Icelandic moss, barley β-glucan, laminaribiose, laminaritriose, laminaritetraose, laminaripentaose, cellotriose, cellobiosyl-β-D-1,3-glucose (G4G3G) were purchased from Megazyme, Ireland. Gentiobiose and laminarin were purchased from Sigma Aldrich. All NMR analyses were performed at Panjab University, Chandigarh, India. All kinetic parameters were performed with GraphPad Prism.

Twelve batches of Ganoderma lucidum (numbered S1 to S12) fruit bodies were collected from different places in China, including S1~S9 from Shouxiangu, Zhejiang Province, S10 from Huangshan, Anhui Province, and S11~S12 from Longquan, Zhejiang Province. The sample information is listed in Table 1. Trifluoroacetic acid (TFA), 1-phenyl-3-methyl-5-pyrazolone (PMP), monosaccharide standards (rhamnose, glucosamine, glucose, mannose, galactose, fucose, arabinose, fructose, xylose, glucuronic acid, and galacturonic acid), lipopolysaccharide (LPS), and polymyxin B were purchased from Sigma-Aldrich (St. Louis, MO, USA). Oligosaccharide standards including laminaribiose, laminaritriose, laminaritetraose, laminaripentaose, and laminarihexaose were purchased from Megazyme (Wicklow, Ireland). Pullulan standards P-5 (M_w = 6300 g/mol) and P-10 (M_w = 9800 g/mol) were purchased from Shodex (Tokyo, Japan). The RAW264.7 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences. DMEM medium and fetal calf serum were bought from Gibco (Grand Island, NY, USA). The mouse TNF-α ELISA kit was bought from Beijing 4A Biotech Co., Ltd. (Beijing, China). All other reagents were analytical grade and produced in China.

To exemplify the function of our enzymatic assay on an environmental sample, we hydrolyzed a sample stepwise and detected the resulting products via HPAEC with pulsed amperometric detection (PAD). The sample extract from Helgoland at April 27, 2017 was first hydrolyzed with 100 nM FbGH30 for 25 min at 37 °C, and then FaGH17A was applied in the same manner. Between each digestion, the reaction was stopped by boiling the sample for 5 min at 100 °C; precipitated protein was removed by filtration through 0.2-µm centrifuge filters (Costar Spin-X; Corning), and an aliquot was taken.
Samples were applied on an ICS-5000+ (Dionex) with electrochemical detection on a gold working electrode and a pH reference electrode (Ag/AgCl) according to Unfried et al. (37 (link)). Separation was attained by using a Dionex CarboPac PA100 analytical column at 35 °C. Glucose (Sigma), laminaribiose, laminaritriose, laminaritetraose, and laminaripentaose (all from Megazyme) were used as reference.