Laminaribiose

Strain Escherichia coli JM109, T4DNA ligase, DNA polymerase PrimeSTAR ^®HS, In-Fusion HD Cloning Plus kit and restriction enzymes were purchased from Dalian BaoBio Co., Ltd. (Dalian, China). The expression host Bacillus subtilis WS11 and the expression vector pBSMμL3 of B. subtilis were constructed in the early works of our laboratory. Agarose gel DNA recovery kit, PCR purification kit and plasmid extraction kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd. (Beijing, China). The coding gene of β-glucosidase TsBgl1 and PCR primers were synthesized by Wuxi Tianlin Biotechnology Co., Ltd. (Wuxi, China). Ampicillin (Amp) and kanamycin (Kan) were purchased from Shanghai Jerry Co., Ltd. (Shanghai, China). The substrates 4-nitrophenyl-β-D-glucopyranoside (pNP-β-Glc), cellobiose, sophorose, gentiobiose and laminaribiose were purchased from Sigma-Aldrich (St. Louis, MO, USA). GOD glucose detection kit was purchased from Shanghai Rongsheng Biotechnology Co., Ltd. (Shanghai, China). Other conventional reagents are purchased from Sinopharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China).

For detection of enzymatic reaction products, high-performance anion exchange column chromatography (HPAEC) with a pulsed amperometoric detector (PAD) equipped with a CarboPac PA10 guard column (4 × 50 mm) and a CarboPac PA10 analytical column (4 × 250 mm; Dionex Co.) was used. Enzymatic reaction was performed by incubation with equivalent volume of rAaBGL1 (20.0 nM) and each substrate in 20 mM sodium acetate buffer (pH 5.0) at 37°C. Reaction mixture was sampled at appropriate time, and added into equal volume of 0.2 M NaOH. Resultant mixtures were subjected to HPAEC-PAD using mobile phase of 100 mM NaOH with 10 mM sodium acetate. Glucose, cellobiose (Wako Pure Chemical Industries, Ltd.), cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, laminaritetraose, laminaripentase (Megazyme), gentiobiose, sophorose (SIGMA-ALDRICH, Co.) were used as standards.

Two strains of Escherichia coli were used in the present work: (i) DH5α for routine molecular biology applications, and (ii) BL21(DE3) Rosetta™ (Novagen) for heterologous proteins production. Both E. coli strains were cultured in LB (BD Difco LB broth) medium supplemented with the appropriate antibiotics (kanamycin [50 μg/mL], chloramphenicol [25 μg/mL]). Streptomyces scabiei 87-22 was routinely cultured at 28°C. Tryptic soy broth (TSB, Sigma-Aldrich, 30 g/L) was used for liquid precultures. The modified TDM (thaxtomin-defined medium (15 (link))), the minimal medium was prepared as described in (10 (link)), and after autoclaving were supplemented with filter-sterilized carbon sources. The substrates used in this study were purchased from Carbosynth (cellobiose, amygdalin, linamarin, xylobiose, laminaribiose, gentiobiose, salicin, arbutin, and syringin), or Sigma-Aldrich (4-nitrophenyl-β-d-glucopyranoside (pNPβG), esculin, cyanin chloride, 4-methylumbelliferyl β-d-glucopyranoside (4-mug), coniferin (abietin), and p-coumaryl alcohol 4-O-glucoside).