Barley β glucan

The substrates beechwood xylan, 4-nitrophenyl β-d-xylopyranoside (pNPX), barley β-glucan, and carboxymethyl cellulose-sodium (CMC-Na) were purchased from Sigma. Soluble and insoluble wheat arabinoxylan were obtained from Megazyme (Ireland). The DNA purification kit, Genome Walking kit, and LA Taq DNA polymerase were purchased from TaKaRa (Japan). T4 DNA ligase and restriction endonucleases were obtained from New England Biolabs (UK). All other chemicals were of analytical grade and commercially available.

Humicola insolens Y1 CGMCC 4573 was cultured routinely in wheat bran medium at 42°C as described previously [13 (link)]. Escherichia coli Trans1-T1 (TransGen, China) was cultivated in Luria-Bertani (LB) medium at 37°C for gene cloning and sequencing. Pichia pastoris GS115 (Invitrogen, USA) was cultivated in yeast peptone dextrose (YPD) medium at 30°C and used for gene expression. The plasmids pEASY-Blunt (TransGen) and pPIC9 (Invitrogen) were used as cloning and expression vectors, respectively. Substrates including barley β-glucan, lichenan, laminarin, birch wood xylan, 4-nitrophenyl β-d-cellobioside (pNPC), 4-nitrophenyl β-d-glucopyranoside (pNPG), and carboxymethyl cellulose sodium salt (CMC-Na) were purchased from Sigma (USA). A DNA purification kit, DNA polymerase, and restriction endonucleases were purchased from TaKaRa (Japan). T4 DNA ligase was purchased from Promega.
Minimal dextrose (MD) medium, buffered glycerol complex (BMGY) medium, and buffered methanol complex (BMMY) medium were prepared according to the manual of the Pichia Expression Kit (Invitrogen).

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.

The endo-β-1,4-glucanase activity was determined by measuring the release of reducing sugar from barley β-glucan (Sigma-Aldrich, St. Louis, MO, USA) with the 3,5-dinitrosalicylic acid (DNS) method [18 ]. The standard reaction mixture, containing 100 μl of appropriately diluted enzyme and 1.0% (w/v) barley β-glucan in 900 μl of McIlvaine buffer (200 mM Na₂HPO₄, 100 mM citric acid, pH 5.0), was incubated in water-bath at 60°C for 10 min and terminated by the addition of 1.5 ml of DNS. After 5-min boiling, the reaction mixture was cooled down to room temperature in ice water. The absorbance was measured at 540 nm. One unit of endo-β-1,4-glucanase activity was defined as the amount of enzyme required to release 1 μmol of glucose per minute.

H. insolens Y1 GCMCC 4573 was routinely cultured in the wheat bran medium45 (link). Escherichia coli Trans1-T1 and vector pEASY-T3 (TransGen, Beijing, China) were used for gene cloning. The gene expression vector and heterologous expression host were pPIC9 and P. pastoris GS115 (Invitrogen, Carlsbad, CA), respectively. The DNA purification kit, restriction endonucleases and LA Taq DNA polymerase were purchased from TaKaRa (Otsu, Japan). T4 DNA ligase and the total RNA isolation system kit were purchased from Promega (Madison, WI). The cDNA synthesis kit was purchased from TransGen. Barley β-glucan, Avicel, 4-nitrophenyl β-d-glucopyranoside (pNPG), 4-nitrophenyl β-d-xylopyranoside (pNPX), 4-nitrophenyl α-l-arabinofuranoside (pNPAf), 4-nitrophenyl α-d-galactopyranoside (pNPGal), 4-nitrophenyl α-l-arabinopyranoside (pNPAb), 4-nitrophenyl β-d-cellobioside (pNPC), disaccharides cellobiose, sophorose and gentibiose and soybean flavones daidzin, genistin and glycitin were all purchased from Sigma-Aldrich. Sodium carboxymethylcellulose (CMC-Na), laminarin and lichenin were obtained from Megazyme (Wicklow, Ireland). All other chemicals used were of analytical grade and commercially available.