Partial Purification and Characterization of Pectinase From Actinomycete : Nocardiopsis Dasnonivelli Isolated From Marine Samples

Abstract Biocatalysis, one of the oldest technologies, is becoming a favorable alternative to chemical processes and a vital part of green technology. This study was to purification and characterization of the pectinase. The extracted enzyme was subjected to partial purification which include ammonium sulphate precipitation, dialysis, sephadex G-100 column chromatography. The cell free supernatant pectinase activity was found to be in the 20% salt saturation fraction. Enzyme activity of pectinase increased from 75.9 ± 0.15U/mL to 81.08 ± 1.06 U/mL at 20% salt saturation fraction. As a result, the 20% ammonium sulphate fraction of crude enzyme extract was further analysed for purification of potential pectinase. Pectinase from the 20% ammonium sulphate crude enzyme fraction was purified by using sephadex-G-100 gel chromatography. From these results, the purified pectinase exhibited 85.83 ± 16 U/ml of enzyme activity. It was observed that the purification of pectinase by sephadex-G-100 gel chromatography increased its activity by 11.51%. The purified pectinase when subjected to SDS PAGE appeared as a single homogenous band with a molecular weight of 40 kDa. Partially purified enzyme was characterized, the enzyme was stable 50C upto 80 min at pH 9. Later the purified 40KDa protein spot of Nocardiopsis dassonvillei S10 was identified as pectinase by Peptide mass fingerprinting. PMF results of the protein spot was matched with Bacillus subtilis Mutant which is having highest score i.e. 63.4.Protein 3D structure models were predicted and validated by the Ramchandran plot assessment and the functional prediction was done by I-TASSER server.