DNA isolation and amplification of ansA1 and ansA3 genes Good quality genomic DNA was isolated

1400 WordsApr 23, 20196 Pages
DNA isolation and amplification of ansA1 and ansA3 genes Good quality genomic DNA was isolated (Sambrook, et al 2002) and bothansA1 and ansA3 genes were amplified by PCR. Clear bands of both the genes showing a size of 1kb were observed under UV transilluminator after agarose gel electrophoresis (Fig 1). Figure 1: PCR amplification of both ansA1 and ansA3 genes: Lane (A): Step up 100bp marker, Lane (B): amplification of ansA1 gene, Lane (C): amplification of ansA3 gene Overexpression and purification The overexpressed recombinant proteins (rBliAI and rBliAIII) of B. licheniformis MTCC 429 were purified to homogeneity by affinity chromatography using Ni-NTA column. Overexpression of recombinant His-tagged asparaginase into E.coli…show more content…
Similarly,while carrying out the characterization of both recombinant (rBliAI and rBliAIII) enzymes from Bacillus licheniformis MTCC 429when expressed in E.coli (DE3) it was observed that recombinant ansA1 enzyme was highly unstable and showed very low activity as compared to recombinant ansA3 enzyme. In earlier reports, E.coli type II L-asparaginase (EcA-II) was found to have a higher affinity for L-asparagine than the type Iisozyme (EcA-I) (Cedar and Schwartz, 1967). Therefore, recombinant ansA3 enzyme was selected for further studies. The results of the purified rBliAIII are summarized in Table 1. Step Total Activity(U) Total Protein(mg/ml) Specific Activity(IU/mg) Purification(Fold) Recovery (%) Crude Extract 3208.3 23.76 135.02 1 100 Ni-NTA 1666.5 4.088 407.65 3.02 52 Table 1: Purification steps of recombinant ansA3 from Bacillus licheniformisMTCC 429 Effect of pH The purified recombinant enzyme was active in a pH range of 7.0-9.0. Maximum asparaginase activity was found at pH 8.0. The enzyme was inactivated at highly acidic and alkaline conditions. The effect of pH on activity is shown in Fig. 5, wherein it can be determined that the enzyme exhibited maximum activity at pH 8. Magdy et al., (2008)showed that purified L-asparaginase II from E.coli W3110 was active in alkaline solutions . At ranges above or below pH 8, the

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