关键词: 侧孢短芽孢杆菌, 天然质粒, 异源表达, 抗菌肽, 抑菌效价

Abstract: The antimicrobial peptide produced by Brevibacillus laterosporus shows efficient, broad-spectrum, and stable antimicrobial activities, which are closely associated with the endogenous plasmid in B. laterosporus S62-9. In order to study the feasibility of heterologous expression of B. laterosporus plasmid with antimicrobial peptide genes, B. laterosporus S62-9 plasmid was transferred into Bacillus subtilis Wb800 by electroporation. The positive transformants were screened and the expression products were characterized. The results showed that strain Z4 showed an antimicrobial activity after fermentation for 6 h, reaching the highest titer (620.96 AU/mL) during 12–20 h. Tricine-SDS-PAGE indicated that B. laterosporus plasmid was successfully expressed in B. subtilis. Reversed-phase high performance liquid chromatography (RP-HPLC) analysis showed that the expression products of strain Z4 and B. laterosporus antimicrobial peptide contained the same substances, which showed broad-spectrum antimicrobial activities with high stability. Both of them displayed strong antimicrobial activities against Gram-positive bacteria, Gram-negative bacteria and fungi, especially against Gram-positive bacteria. About 90% and 81% of the titer was retained after 20 min at 121 ℃ and after 60 min at 100 ℃, respectively. The antimicrobial activity showed a slight change in the pH range of 2–12. However, the antimicrobial peptide was more sensitive to proteases and the titer was reduced by about 40% after treatment with trypsin or proteinase K, and was also reduced after treatment with three other proteases. The establishment of strain Z4 may lay a foundation for the industrial production of antimicrobial peptide in the future, and provide evidence for positioning the B. laterosporus antimicrobial peptide gene on plasmid.

Key words: Brevibacillus laterosporus, natural plasmid, heterologous expression, antimicrobial peptide, inhibitory titers