Improvement of an Acid Phosphatase/DHAP‐Dependent Aldolase Cascade Reaction by Using Directed Evolution

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.

Phosphorylation by Alkaline Phosphatase: Immobilization and Synthetic Potential

“…That no hydrolysis of DHAP occurs is probably caused by inactivation of the AP by the phosphate formed. The results obtained from these experiments agree with previous studies performed with acid phosphatases PhoN-Se and PhoN-Sf in which respectively 1.6 and 3 mM of DHAP were obtained at pH 4, using the same concentrations of PP i and DHA (van Herk, Hartog, Schoemaker et al, 2006;van Herk, Hartog, Babich et al, 2009). In contrast the acid phosphatases are not inhibited by phosphate formed and phosphorylation of DHA was rapidly followed by dephosphorylation of DHAP.…”

“…typhimurium (PhoN-Se), and Morganella morganii have been widely used in the regioselectively phosphorylation of nucleosides such as inosine and guanosine to the corresponding 5'-phosphate derivatives (5'-IMP and 5'-GMP) used as taste enhancers (umami) (Asano, Mihara, & Yamada, 1999a;Asano et al, 1999b;Low & Saltiel, 1988;Mihara, Utagawa, Yamada, & Asano, 2000;Mihara, Utagawa, Yamada, & Asano, 2001;Tanaka et al, 2003), of glucose to glucose-6-phosphate, and of many other primary alcohols, such as glycerol and DHA (Babich et al, 2011;Tanaka et al, 2003;van Herk, Hartog, van der Burg et al, 2005;van Herk, Hartog, Schoemaker et al, 2006;van Herk, Hartog, Babich et al, 2009). …”

“…These phosphorylated alcohols are key intermediates in two enzymatic cascade reactions leading to the synthesis of carbohydrates (Figure 1) (Babich et al, 2011;van Herk, Hartog, Schoemaker et al, 2006;van Herk, Hartog, Babich et al, 2009 Vol. 5, No.…”

One‐Pot Cascade Reactions using Fructose‐6‐phosphate Aldolase: Efficient Synthesis of D‐Arabinose 5‐Phosphate, D‐Fructose 6‐Phosphate and Analogues

“…Enzymatic methods ensure mild reaction conditions and furthermore, tedious protection and deprotection of functional groups often resulting in low yields can be circumvented. [5] For the preparative synthesis of monophosphorylated sugars two approaches are mainly found in the literature depending on the enzymes used: (i) by selective phosphorylation of an alcohol function and (ii) by C-C bond formation. The first method is usually completed through the use of kinases albeit they often display narrow substrate specificity.…”

“…[6] As an example, D-arabinose-5-phosphate, an important metabolite involved in the biosynthetic pathway of the outer membrane component lipopolysaccharide (LPS), was prepared via a kinase catalysed reaction. [7] Direct phosphorylation of a hydroxyl group using phosphatases or phosphotransferases and inorganic pyrophosphate have also been described by Wever et al [5] In such process the key intermediate in the mechanism is an activated phosphorylated enzyme intermediate that may react with a nucleophile like for example primary alcohol function to yield a phosphorylated product. Water being a competitive nucleophile, the intermediate may also be hydrolysed, resulting in formation of free phosphate.…”