Bacillus sp. N16-5 Cultivation and Mutant Construction

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.