Heterologous expression of CYP102A5 variant from Bacillus cereus CYPPB-1: Validation of model for predicting drug metabolism of human P450 probe substrates.

CYP102A5 variant (ADL27534) from isolated Bacillus cereus CYPPB-1 was heterologously expressed in Escherichia coli Top 10 cells. Comparative sequence analysis of purified CYP102A5 variant with respect to reported CYP102A5 (AAP10153) from Bacillus cereus ATCC 14579 revealed amino acid sequence changes at positions P245S and M318I of heme domain. The binding affinities of 15 selected human P450 probe substrates towards isolated CYP102A5 were analyzed in silico using a homology model together with molecular docking techniques to predict the human drug metabolism. In vitro analysis suggested that the purified CYP102A5 metabolizes typical substrates of human CYP2C9, CYP2D6, CYP2E1, and CYP3A4, such as coumarin, propranolol, aniline, chlorzoxazone, p-nitrophenol, and nifedipine. The calculated K M values for propranolol, chloroxazone, coumarin, aniline, and 4-nitrophenol were calculated to be 0.962 ± 0.041, 1.254 ± 0.057, 2.859 ± 0.083, 2.732 ± 0.106, and 2.528 ± 0.11 mM, respectively. Importantly, taking a ChemScore cutoff value of -31 kJ/mol, substrate binding at active site and in vitro activity as the distinguishing lines between "substrates" and "nonsubstrates" revealed one false-positive and one false-negative results out of the 15 compounds examined. This is the first report on validation of CYP102A family homology model for in silico prediction of human drug metabolism.

Purification, characterization and production optimization of a vibriocin produced by mangrove associated Vibrio parahaemolyticus.

OBJECTIVE: To identify a potential bacterium which produces antimicrobial peptide (vibriocin), and its purification, characterization and production optimization. The bacteria subjected in the study were isolated from a highly competitive ecological niche of mangrove ecosystem. METHODS: The bacterium was characterized by phenotype besides 16S rRNA gene sequence analysis. The antibacterial activity was recognised by using agar well diffusion method. The vibriocin was purified using ammonium sulphate precipitation, butanol extraction, gel filtration chromatography, ion-exchange chromatography and subsequently, by HPLC. Molecular weight of the substance identified in SDS-PAGE. Production optimization performed according to Taguchi's mathematical model using 6 different nutritional parameters as variables. RESULTS: The objective bacterium was identified as Vibrio parahaemolyticus. The vibriocin showed 18 KDa of molecular mass with mono peptide in nature and highest activity against pathogenic Vibrio harveyi. The peptide act stable in a wide range of pH, temperature, UV radiation, solvents and chemicals utilized. An overall ∼20% of vibriocin production was improved, and was noticed that NaCl and agitation speed played a vital role in secretion of vibriocin. CONCLUSION: The vibriocin identified here would be an effective alternative for chemically synthesized drugs for the management of Vibrio infections in mariculture industry.