In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp), and the product was cloned into a pGEM-T vector. The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3). The recombinant E. coli was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax) and Michaelis constant (Km) were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS).
Even purified enzyme preparations are often heterogeneous. For example, preparations of aspartate aminotransferase or cytochrome oxidase can consist of several different forms of the enzyme. For this reason we consider how different the kinetics of the reactions catalysed by a mixture of forms of an enzyme must be to provide some indication of the characteristics of the species present. Based on the standard Michaelis-Menten model, we show that if the Michaelis constants (Km) of two isoforms differ by a factor of at least 20 the steady-state kinetics can be used to characterise the mixture. However, even if heterogeneity is reflected in the kinetic data, the proportions of the different forms of the enzyme cannot be estimated from the kinetic data alone. Consequently, the heterogeneity of enzyme preparations is rarely reflected in measurements of their steady-state kinetics unless the species present have significantly different kinetic properties. This has two implications: (1) it is difficult, but not impossible, to detect molecular heterogeneity using kinetic data and (2) even when it is possible, a considerable quantity of high quality data is required.
Current re-emergence of Nipah virus (NiV) in India caused 11 deaths so far and many patients were kept in quarantine. A thorough study of previous outbreaks occurred in Malaysia, Bangladesh and India represents cases with high rate of fatality due to acute encephalitis. Our work involves genome analysis of NiV for prediction of miRNAs and their targeted genes in human in order to understand encephalitis origin. Ab-intio program-VMir was used for initial screening of genome, obtained nine pre-miRNAs was analyzed by ViralMir to check for any pseudo pre-miRNAs. Eighteen functional mature miRNAs were extracted from pre-miRNAs by using Mature-Bayes tool, which targets 669 genes in human genome as retrieved by miRDB. Gene ontology terms by PANTHER provide important pathways in which target genes were involved like Axon guidance, T cell activation, and nicotinic acetylcholine receptor signaling. Significant outcome was obtained after NCBI Gene and OMIM database mining and literature search for predicted target genes. TLR3, TJP1, NOTCH2, FHL1, and GRIA3 target genes obtained showed their involvement in host defense, blood brain barrier, neurogenesis, mental retardation and encephalitis. To conclude, we predicted significant genes in human that can be inhibited by miRNAs of NiV and results in etiology of encephalitis.