Prokaryotic Expression, Purification and Activity Determination of Sortase A Enzyme Mutant
In this study,the SrtA△N25,a transpeptidase derived from Staphylococcus aureus,and its mutants(m5SrtA△N59 and m9SrtA△N59) were expressed in pET28a expression system,purified via Ni-Sepharose and cation-exchangechromatography subsequently.The enzyme activities,kinetic parameters and optimal reaction conditions of these enzymeswere assessed on FRET-based platform,and effects of different biochemical reagents were evaluated on SrtA activity.Asa result,SrtA△N25,m5SrtA△N59 and m9SrtA△N59 were expressed as a soluble form in Escherichia coli with a high yield andmaintained enzyme activities.Finally,the Kcat/Km values of m5SrtA△N59 and m9SrtA△N59 respectively increased 68-fold and5 544-fold compared to that of the SrtA△N25,suggesting the catalytic efficiency of m9SrtA△N59 was improved significantly.The optimum reaction temperature of m9SrtA△N59was 30-37 ℃ and the optimum pH value was 6.0-7.0.Adding reducingagents such as tris-(2-carboxyethyl) phosphine (TCEP) and dithiothreitol (DTT) to the reaction system was helpful forenhancing the catalytic activity of SrtA enzyme.
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