Integrated approach for purification of uricase and protease from Bacillus licheniformis by TPP and IEC.

Abstract

In order to explore the separation and purification methods of uricase and alkaline protease crude enzyme extracts, a combinatorial approach of Three Phase Partitioning (TPP) and Ion-Exchange Chromatography (IEC) was studied. TPP alone was able to separate and purify uricase and alkaline protease enzymes by 5 fold and 2.7 fold, respectively. Further application of ion-exchange chromatography purified enzymes with 99.99% purity in the form of single peaks on a chromatogram. The optimum TPP parameters for simultaneous separation and purification were 50% ammonium sulfate concentration, crude extract to solvent ratio of 1:1, and pH of 8.5. Ion exchange chromatography was performed on a fully automated AKTA start system equipped with a conductivity detector, UV detector, fraction collector, and buffer reservoirs used for isocratic or gradient elution profiles of the proteins under study. Successful purification of enzymes, including their molecular weight, was confirmed with SDS PAGE analysis. Furthermore, the uricase sequence from Bacillus licheniformis was also corroborated to be 87.6% homologous to uricase of B. subtilis using the bioinformatics tool BLASTp (Basic Local Alignment Search Tool for Protein), wherein it compares sequence similarity based on protein or nucleotide sequence. It should be emphasized that this study is the first report for tandem enzyme purification of two enzymes using an automated IEC - AKTA start system where partial purification of enzymes was carried out using TPP.