Enhancement of antibacterial activity of sinigrin-capped silver nanoparticles in combination with myrosinase

Abstract

Green synthesis of silver nanoparticles (AgNPs) is a widely accepted approach due to its ecofriendly use of non-toxic materials. AgNPs are extensively used in the agriculture and medicine industry as antioxidant, antibacterial, and antifungal agents. In this study, AgNPs were synthesized using phytochemical sinigrin as both reducing and capping agent. Sinigrin present in cruciferous vegetables can be hydrolyzed by endogenous myrosinase (MYR) to produce allyl isothiocyanate (AITC) which has reported antibacterial properties. We hypothesized that sinigrin-capped AgNPs (SIN-AgNPs) will have enhanced antibacterial activity in the presence of MYR enzyme due to the release of AITC from hydrolysis of sinigrin. The formation of SIN-AgNPs was confirmed by UV-Vis absorption spectra which exhibited a peak around 405 nm. The particle size was determined to be ~20 nm using dynamic light scattering. The size was also confirmed through transmission electron microscopy (TEM) imaging with morphology close to spherical shape. Fourier transform infrared analysis confirmed the functional groups of sinigrin capped on the surface of AgNPs. Myrosinase gene (TGG1) sequence was cloned in pET-20b(+) plasmid and expressed in BL21(DE3) E. coli competent cells. The collected bacterial lysate was purified by immobilized metal affinity chromatography to obtain MYR protein which was characterized by SDS-PAGE, Western blot, and enzyme activity. The antibacterial efficacy was evaluated against E. coli using disc diffusion assay and growth kinetics study. Our results showed that SIN-AgNPs in combination with MYR protein exhibited ⁓25% more zone of inhibition by disk diffusion assay at a concentration of 25 µg/ml and inhibited bacterial growth for a longer time in the range of 0.5–1 µg/ml in growth kinetic assay.