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Improvement of an Acid Phosphatase/DHAP‐Dependent Aldolase Cascade Reaction by Using Directed Evolution
Author(s) -
van Herk Teunie,
Hartog Aloysius F.,
Babich Lara,
Schoemaker Hans E.,
Wever Ron
Publication year - 2009
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200900102
Subject(s) - dhap , aldolase a , salmonella enterica , phosphatase , acid phosphatase , dihydroxyacetone phosphate , shigella flexneri , mutant , biochemistry , salmonella , fructose bisphosphate aldolase , enzyme , chemistry , dihydroxyacetone , active site , wild type , biology , microbiology and biotechnology , escherichia coli , bacteria , gene , genetics , glycerol
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.