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Improvement of an Acid Phosphatase/DHAP‐Dependent Aldolase Cascade Reaction by Using Directed Evolution
Authors:Teunie van Herk Dr.  Aloysius F. Hartog  Lara Babich  Hans E. Schoemaker Prof.  Ron Wever Prof.
Affiliation:Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 129, 1018WS Amsterdam (The Netherlands), Fax: (+31)?20‐525‐5670
Abstract:To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild‐type enzyme at pH 6.0. This variant is 17 times more active than the wild‐type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.
Keywords:acid phosphatases  aldol reaction  cascade reactions  dihydroxyacetone phosphate  directed evolution  high‐throughput screening
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