Studies on metal tolerant bacteria and applications of porphyrin derivatives

Abstract

Metal resistant property of an isolate from Brassica oleracea var. capitata (Cabbage) was studied for the first time. Among the isolates, the strain PGSV4207 showed heavy metal tolerance at high concentrations. This potent isolate was identified as Pseudomonas aeruginosa by morphological observation, biochemical and physiological characterization and was further confirmed by 16S rDNA sequence analysis (NCBI Accession no. EU732606). The uptake of heavy metals like cadmium, lead, nickel and zinc was confirmed by atomic absorption spectroscopic studies (AAS). We have analysed the isolate PGSV4207 for lipase production and the protein was purified by ammonium sulphate precipitation followed by hydrophobic interaction chromatography. We have used tetrakis-(4-sulphanato)-phenyl porphyrin (TPPS) to study the aggregation of Candida rugosa lipase and P.aeruginosa lipase in Native gel. This method overcomes protein aggregation that occurs when native PAGE is used to study these proteins. We have developed a method for in-gel staining of proteins using tetrakis (4-sulphonato phenyl) porphyrin (TPPS). This method is inexpensive, easy to perform and also aids in efficient recovery of protein from Native-PAGE for further analysis. Advantage of TPPS in protein identification is that it does not involve the problem of post-electrophoretic techniques. The substrate triacetin is broken down into acetic acid by the lipolytic activity and the drop in pH brings changes TPPS from green to red. This change can be visualized by naked eye as well as by spectrophotometric analysis. Live-cell imaging requires the use of non-cytotoxic dyes at low concentrations at non-harmful visible wavelengths. TPPS was used as a stain to visualize Pseudomonas aeruginosa and Bacillus cereus using fluorescent microscopy. TPPS was effectively used to analyse two strains of bacteria. The variation of brightness with varying concentrations of TPPS was studied and Bacillus was found to have consistently higher brightness. newline newline newline