Lichenan

The acidothermophilic Alicyclobacillus sp. A4 (whole genome sequenced) isolated from the hot spring water was stored in our laboratory [1 (link)]. GH3 β-glucosidase Bgl3A derived from Talaromyces leycettanus JCM12802 [376] was used as the reference. Ni²⁺-affinity beads from Suzhou Beaver Biomedical Engineering (China), and glucose oxidation (GOD-POD) kit from Beijing Leadman (China) were purchased. Substrates of p-nitrophenyl β-d-glucopyranoside (pNPG), p-nitrophenyl α-l-arabinofuranoside (pNPAf), p-nitrophenyl β-d-xylopyranoside (pNPX), barley β-glucan, lichenan, Avicel, and standard samples of daidzein, glycitein and genistein from Sigma-Aldrich (USA), cellobiose to cellohexose, laminaritetraose and maltose from Megazyme (Ireland), and daidzin from Tokyo Chemical Industry were obtained. The soybean meal was purchased from local market. All the other reagents were of analytical grade and commercially available.

Talaromyces leycettanus JCM12802 was purchased from the Japan Collection of Microorganisms RIKEN BioResource Center (Tsukuba, Japan). E. coli Trans1-T1, vector pEASY-T3, the DNA purification kit, LA Taq DNA polymerase and Genome Walking kit were purchased from TaKaRa (Tsu, Japan). The RNA isolation system kit was purchased from Promega (Madison, WI, USA). Restriction endonucleases, T₄ DNA ligase and Endo H (endo-β-N-acetylglucosaminidase H) were purchased from New England Biolabs (Ipswich, MA). The DNA isolation kit and pfu DNA polymerase were purchased from Tiangen (Beijing, China). The substrates barley β-glucan, lichenan, laminarin, CMC-Na, birchwood xylan, Avicel, pNPG, pNPC and carob bean gum were purchased from Sigma-Aldrich (St. Louis, MO). The plasmid pPIC9 and P. pastoris GS115 (Invitrogen, Carlsbad, CA) were used as the gene expression vector and expression host strain, respectively. The low-molecular-weight calibration kit was purchased from GE Healthcare (Pittsburgh, PA). Minimal dextrose medium (MD), buffered glycerol-complex medium (BMGY) and buffered methanol-complex medium (BMMY) were prepared as described in the instructions of the Pichia Expression Kit (Invitrogen). All chemicals were of analytical grade and commercially available.

The substrate specificity of AC2aCel5A had previously been determined using Azurine-Crosslinked Polysaccharide (AZCL) substrates, along with activity on insoluble cellulose substrates7 (link). In this study, further characterization of the enzymatic activity was performed using the soluble β-(1,4) linked glucan substrates carboxymethylcelullose (CMC) (Sigma-Aldrich), barley β-glucan (Megazyme), tamarind xyloglucan (Megazyme), lichenan (Sigma-Aldrich), and Birchwood Xylan (Carl Roth). The standard reaction using CMC contained 20 mM BisTris buffer pH 6.5, 25 nM enzyme, 20 mM CaCl₂, and 10 mg/ml substrate in a total volume of 200 μl. Enzyme was added to pre-heated assay mixtures and the reactions were incubated at 40 °C, with 900 rpm vertical shaking. 100 μl sample was taken after 10 minutes, and added to 100 μL DNS reagent40 . The amount of reducing ends released were determined as glucose equivalents using the DNS reducing-end assay and a glucose standard curve. Assays for activity on barley β-glucan, lichenan, xylan and xyloglucan were performed with 0.5% substrate (w/v), and 10, 10, 200 and 200 nM enzyme load, respectively. A Unit of enzyme activity was defined as the amount of enzyme releasing one μmol of glucose equivalents per minute.

If not stated otherwise, all chemicals were purchased from VWR (Darmstadt, Germany). Lichenan was obtained from Sigma-Aldrich (Deisenhofen, Germany).
The following antibodies were used: β-actin (AC-15), mouse monoclonal (1:4000, 1 h at RT) from Sigma-Aldrich; KRT1 (1:200,000 for 2 h at RT), KRT10 (1:10,000 at 4 °C over night) and IVL (1:500,000 at 4 °C at RT), and rabbit monoclonals from Abcam; Rabbit anti-Mouse IgG HRP conjugate from Jackson ImmunoResearch; and Rabbit anti-Mouse IgG HRP (1:10,000 for 45 min at RT) from Jackson Immuno Research.
NMR spectra were recorded on an Agilent VNMRS 600 (Agilent Technologies, CA, USA). ¹H and ¹³C-NMR measurements were obtained at 80 °C at 600 MHz and 150 MHz, respectively. ¹H-¹³C-HSQC-NMR spectra were recorded at 20 °C. Data analysis was achieved with MestRneNova software version 10.0.0-14381. Sample concentration: 5 mg/mL D₂O (Uvasol^®, 99.8%, Merck, Darmstadt, Germany) and DMSO-D6 was added for referencing (δ 3.330 ppm).
Gel permeation chromatography was performed on a low pressure Sepharose^®6 stationary phase (GE Healthcare, Freiburg, Germany) using standard dextrans (Sigma, Deisenhofen, Germany) for calibration. Each fraction was tested on the carbohydrate content accordingly [28 (link)].