International Journal of Pharma and Bio Sciences
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10.22376/ijpbs.2019.10.1.p1-12
Volume 7 Issue 4
2016 (October - December)
Site-directed mutagenesis on alkaline phosphatasefrom pseudomonas aeruginosapao1 for enhanced activity
Alkaline phosphatase (ALP) is a hydrolase enzyme known for removing phosphate groups from mono and di-esters and widely used in bioremediation of heavy metals. For effective bioremediation higher turnover rate is required so that the product inorganic phosphate precipitates metals more rapidly. In this paper, the phoA gene from lessThan i greaterThan Pseudomonas aeruginosa lessThan /i greaterThan PAO1 was cloned, over-expressed in lessThan i greaterThan E.coli lessThan /i greaterThan BL21 (DE3) and further purified and characterized. The molecular mass of recombinant enzyme was determined on SDS-PAGE and was found to be 52 kDa. Mutations were performed at the active site residues of the enzyme. Maximum catalytic activity for wild-type enzyme was observed at 45-50°C, pH 8.0 with K lessThan sub greaterThan m lessThan /sub greaterThan of 75 µM and k lessThan sub greaterThan cat lessThan /sub greaterThan 113 s lessThan sup greaterThan -1 lessThan /sup greaterThan on the substrate p-nitrophenol phosphate. Interestingly, the H412R mutant showed 8-fold increase in activity and 6-fold increase in k lessThan sub greaterThan cat lessThan /sub greaterThan /K lessThan sub greaterThan m lessThan /sub greaterThan in the presence of 1 mM cobalt. This enhanced enzyme activity after mutation proves effective bioremediation results.
UMA SELVARAJ, THIRUMALAI MUTHUKUMARESAN, GAYATHRI VIJAYENDRAN, SENTHIL KUMAR DEVAN, P. VENU-BABU AND RICHARD THILAGARAJ WILSON
Alkaline phosphatase, Pseudomonas aeruginosa, site-directed mutagenesis, H412R mutant, cobalt.
877-884