Fig. 4
Detection of β-1,3-1,4-glucanase in the culture supernatant of Bacillus sp. GFP-2 and conformation of its antimicrobial activities. A The crude proteins in extracellular metabolites of Bacillus sp. GFP-2 were extracted by ammonium sulfate precipitation method from the culture supernatant. Then 200 μl of Na3PO4 buffer (a), or the crude proteins (b) was inoculated into the β-1,3-1,4-glucanase identification agar plates (see “Materials and methods”) and the plates were incubated at 37 °C for 12 h. B DNA sequence encoding β-1,3-1,4-glucanase was cloned from GFP-2 genome into the expression construct pET-28a(+) by PCR, which was transformed into E. coli BL21(DE3) to express the recombinant β-1,3-1,4-glucanase with (line 2) or without (line 1) the stimulation of IPTG. The recombinant protein was further purified through Ni-chelating affinity chromatography and analyzed by Western blot and Coomassie blue staining. C 200 μl of elution buffer (a), supernatant of engineered bacteria lysate (b), or protein elution solutions (c and d) were inoculated into the β-1,3-1,4-glucanase identification agar plates (see “Materials and methods”) and the plates were incubated at 37 °C for 12 h. D Activated Bacillus subtilis WB800 N (left) and Escherichia coli BL21 (right) were inoculated as target bacteria into LB agar plates. Sterilized oxford cups were put on pre-coating LB agar plates and 250 μl of LB medium (a, negative control), ampicillin (1 mg/ml) (b, positive control), or purified recombinant β-1,3-1,4-glucanase were added onto the plates, and the plates were incubated at 37 °C for 12 h. E GFP-2 culture media (10 μl, line 1) and the recombinant β-1,3-1,4-glucanase (10 μl, 0.913 μg/μl) (line 2) (positive control, see “Materials and methods” for details) were analyzed by SDS-PAGE and western blot using the rabbit serum containing antibody against β-1,3-1,4-glucanase (see “Materials and methods” for details)