Galactosyl mannotriose

Celluclast 1.5 L, Cellic CTec 2, Viscozyme L (manufactured by Novozymes Corp.), oat-spelt xylan (95590), Amberlite XAD4, and Amberlite IRA958 were purchased from Sigma (St. Louis, MO, USA). The T. reesei β-mannanase (TrMan5A) was obtained as previously described by Hägglund et al. [73 (link)]. AcGGM from thermomechanical pulping (TMP-AcGGM) was obtained as described in Andersson et al. [40 (link)]. Mannobiose (M2), mannotriose (M3), mannotetraose (M4), mannopentaose (M5), mannohexaose (M6), galactosyl-mannotriose (GM3), di-galactosyl-mannopentaose (G2M5), para-nitrophenyl (pNP)-α-D-galactopyranoside, pNP-β-D-glucopyranoside, pNP-β-D-mannopyranoside, pNP-β-D-xylopyranoside and low viscosity locust bean gum (lvLBG) were purchased from Megazyme (Bray, Ireland). Acetone and pullulan molecular weight standards were procured from Merck (Darmstadt, Germany).

Table 1 lists the plasmids and bacterial strains used in this study. Escherichia coli DH5α and BL21(DE3) were grown in LB medium at 37°C and 50 μg/mL kanamycin was added to select plasmid transformants. Bacillus sp. N16-5 was cultivated in Horikoshi-II medium [19 ] in aerobic conditions at 37°C in shaken flasks. SA5 medium [20 (link)] was used in protoplast regeneration of Bacillus sp. N16-5 and neutral complex medium (NCM) [21 (link)] was used in deletion mutant construction.
All substrates (mannobiose, mannotriose, mannotetraose, mannopentose, galactosyl-mannotriose, xylotriose, and xylotetraose) were purchased from Megazyme (Wicklow, Ireland). Locust bean gum was purchased from Sigma Chemical Co. (St. Louis, Mo, USA). All other chemicals were commercially available and of analytical grade.