ABSTRACT
FAD Synthetase (FADS) [EC 2.7.7.2], the second enzyme in flavin cofactor biosynthetic pathway converts FMN to FAD, plays an important role in many redox reactions. Neurospora crassa FADS (NcFADS) was cloned and overexpressed in E. coli cells. Recombinant NcFADS was purified in high yields of â¼8 mg per liter of bacterial culture using a single step glutathione sepharose affinity chromatography. SDS-PAGE and MALDI-MS revealed that NcFADS has a molecular mass of â¼31 kDa. Enzyme kinetic analysis monitored by reverse phase HPLC demonstrate a specific activity and kcat of 1356 nmol/min/mg and 0.69sec-1 respectively. Steady state kinetic analysis of NcFADS exhibited a Km of NcFADS for FMN is 2.7 µM and for MgATP-2 is 88.7 µM. Isothermal titration calorimetry experiments showed that the recombinant protein binds to the substrates with apparent Kd of 20.8 µM for FMN and 16.6 µM for MgATP-2. Biophysical characterization using intrinsic fluorescence suggests that the enzyme is in folded conformation. Far-UV CD data suggest that the backbone of the enzyme is predominantly in a helical conformation. Differential scanning calorimetry data shows that the Tm is 53 °C ± 1. This is the first report on cloning, purification and characterization of FADS from N. crassa. The specific activity of NcFADS is the highest than any of the reported FADS from any other source. The results obtained in this study is expected to pave way for intensive research aimed to understand the molecular basis for the extraordinarily high turnover rate of NcFADS.
ABSTRACT
Eco-friendly synthesis of the silver nanoparticles (AgNPs) has a number of advantages like simplicity, biocompatibility, low toxicity in nature over their physical and chemical methods. In the present study, the authors report biosynthesized AgNPs using the root extract of the perennial plant 'Spiny gourd' (Momordica dioica) and investigated their anti-bacterial application with mechanistic approaches. Different biophysical techniques such as UV-Vis spectroscopy, FTIR, XRD, TEM, SAED, and DLS were employed for AgNPs characterization. The synthesized AgNPs were polydispersed, crystalline in nature, with anionic surface (-22.3 mV), spherical in shape with an average size of 13.2 nm. In addition, the AgNPs were stable in room temperature and in different biological buffers. The anti-bacterial activities of AgNPs were studied with respect to the pathogens such as Bacillus subtilis, Staphylococcus aureus (Gram-positive), Pseudomonas aeruginosa, Escherichia coli, Klebsiella planticola (Gram-negative), and Candida albicans. Also, mechanistic studies of AgNPs such as protein leakage assay, nucleic acid leakage assay, ATP leakage assay, ROS accumulation, determination of biofilm degrading activity, measurement of potassium, showing that the synthesized AgNPs are capable of containing a potential application in the antimicrobial therapeutic agents and the pharmaceutical industry.
