Journal
CHEMBIOCHEM
Volume 10, Issue 13, Pages 2230-2235Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.200900102
Keywords
acid phosphatases; aldol reaction; cascade reactions; dihydroxyacetone phosphate; directed evolution; high-throughput screening
Funding
- Dutch National Research School Combination Catalysis (NRSC-Catalysis).
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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.
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