Antibacterial efficacy of recombinant Siganus oramin L-amino acid oxidase expressed in Pichia pastoris

Abstract

Siganus oraminl-amino acid oxidase is a novel natural protein (named SR-LAAO) isolated from serum of the rabbitfish (S. oramin), which showed antibacterial activity against both Gram-positive and Gram-negative bacteria and had a lethal effect on the parasites Cryptocaryon irritans, Trypanosoma brucei brucei and Ichthyophthirius multifiliis. In order to test whether recombinant SR-LAAO (rSR-LAAO) produced by the eukaryotic expression system also has antimicrobial activity, the yeast Pichia pastoris was used as the expression host to obtain rSR-LAAO in vitro. Crude rSR-LAAO produced by P. pastoris integrated with the SR-LAAO gene had antibacterial activity against both Gram-positive and Gram-negative bacteria as shown by inhibition zone assay of the antibacterial spectrum on agar plates. The average diameter of the inhibition zone of crude rSR-LAAO against the Gram-positive bacteria Staphylococcus aureus and Streptococcus agalactiae was 1.040 ± 0.045 cm and 1.209 ± 0.085 cm, respectively. For the Gram-negative bacteria Aeromonas sobria, Escherichia coli, Vibrio alginolyticus, Vibrio cholera and Photobacterium damselae subsp. piscicida, the average diameter of inhibition zone was 1.291 ± 0.089 cm, 0.943 ± 0.061 cm, 0.756 ± 0.057 cm, 0.834 ± 0.023 cm and 1.211 ± 0.026 cm, respectively. These results were obtained at the logarithmic growth phase of S. agalactiae and A. sobria cell suspensions after incubation with 0.5 mg/mL crude rSR-LAAO for 24 h. The final bacterial growth rate was decreased significantly. The relative inhibition rate can reach 50% compared to crude products from P. pastoris integrated with an empty vector at the same concentration of protein. The antimicrobial activity of crude rSR-LAAO was likely associated with H2O2 formation, because its inhibition zones were disturbed significantly by catalase. Scanning electron microscopy results showed crude rSR-LAAO-treated bacterial surfaces became rough and particles were attached, cell walls were retracted and cell membranes were ruptured. Together, the results of this study indicated rSR-LAAO from the P. pastoris expression system is a potential antibiotic for application as a therapeutic agent against bacterial diseases.

Keywords: Antibacterial efficacy; Pichia pastoris; Recombinant l-amino acid oxidase; Siganus oramin.