5 years ago

Investigation of acid phosphatase variants for the synthesis of phosphate monoesters

Investigation of acid phosphatase variants for the synthesis of phosphate monoesters
Gábor Tasnádi, Kai Baldenius, Michaela Zechner, Kurt Faber, Klaus Ditrich, Mélanie Hall
The major drawback of using phosphatases for transphosphorylation reactions lies in product depletion caused by the natural hydrolytic activity of the enzymes. Variants of PhoC-Mm from Morganella morganii and NSAP-Eb from Escherichia blattae were studied for their ability to maintain a high product level in the transphosphorylation of various primary alcohols. A single amino acid exchange delivered phosphatase variant PhoC-Mm G92D, which was able to catalyze the phosphorylation of primary alcohols without any major hydrolysis of the formed phosphate esters. The mutation mostly improved the affinity of the enzyme for alcohols, while rate constants of transphosphorylation and hydrolysis were decreased, overall resulting in a superior catalytic efficiency in transphosphorylation compared to hydrolysis. The presence of residual substrate alcohol at a given concentration was crucial to suppress phosphate ester hydrolysis. The present work extends the synthetic applicability of phosphatase variants beyond the previously reported nucleosides and allows preparative-scale production of various primary phosphate esters (yields up to 42%) with high enzyme productivity (TONs up to ∼66,000). Biotechnol. Bioeng. 2017;9999: 1–9. © 2017 Wiley Periodicals, Inc. The phosphorylation of a range of alcohols using acid phosphatases from Morganella morganii and Escherichia blattae at the expense of inorganic pyrophosphate is reported. Two enzyme mutants exhibited reduced hydrolytic activity on the formed products and enabled the synthesis of phosphate monoesters on gram-scale. The present work represents a general and facile method to maintain high product level in phosphatase-catalyzed transphosphorylation processes.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/bit.26352

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