Preparative‐Scale Enzymatic Synthesis of rac‐Glycerol‐1‐phosphate from Crude Glycerol Using Acid Phosphatases and Phosphate

Abstract

Glycerol is a byproduct of biodiesel production and is generated in large amounts, which has resulted in an increased interest in its valorization. In addition to its use as an energy source directly, the chemical modification of glycerol may result in value‐added derivatives. Herein, acid phosphatases employed in the synthetic mode were evaluated for the enzymatic phosphorylation of glycerol. Nonspecific acid phosphatases could tolerate glycerol concentrations up to 80 wt % and pyrophosphate concentrations up to 20 wt % and led to product titers up to 167 g L−1 in a kinetic approach. In the complementary thermodynamic approach, phytases were able to condense glycerol and inorganic monophosphate directly. This unexpected behavior enabled the simple and cost‐effective production of rac‐glycerol‐1‐phosphate from crude glycerol obtained from a biodiesel plant. A preparative‐scale synthesis on a 100 mL‐scale resulted in the production of 16.6 g of rac‐glycerol‐1‐phosphate with a reasonable purity (≈75 %).

On the donor substrate dependence of group‐transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase‐catalyzed transglycosylation

“…The influence of the donor substrate is considerably more pronounced than in sucrose phosphorylase. Other phosphatases behave similarly (Han & Coleman, 1995;Tasnádi et al, 2020;van Herk, Hartog, van der Burg & Wever, 2005). Similar behavior has been noted for select esterases/lipases and proteases/N-acylases (Adlercreutz, 2017;Marsden et al, 2020;Müller et al, 2020).…”

“…Glycoside hydrolases (Adlercreutz, 2017;Seibel et al, 2006;Vera et al, 2020;Zeuner, Jers, Mikkelsen, & Meyer, 2014), lipases (Adlercreutz, 2017;Subileau et al, 2017), amidases (Bruggink, Roos, & de Vroom, 1998;Giordano et al, 2006;Gololobov et al, 1990;Sio & Quax, 2004;Sklyarenko, El'darov, Kurochkina, & Yarotsky, 2015), and phosphatases (Ishikawa et al, 2002;Tasnádi, Staśko, Ditrich, Hall, & Faber, 2020; are industrial enzymes used in these synthetic processes. Mechanistically, the enzymatic reactions proceed in two catalytic steps via a covalent enzyme intermediate (Gololobov, Borisov, Belikov, & Švedas, 1988;Kasche, 1986;Marsden et al, 2020;Seibel et al, 2006;Zeuner et al, 2014).…”

Key advances in biocatalytic phosphorylations in the last two decades: Biocatalytic syntheses in vitro and biotransformations in vivo (in humans)

“…[58] F i g u r e 4 Selected phosphatase-catalyzed O-phosphorylation reactions for the synthesis of phosphorylated building blocks, such as monophosphorylated alcohols, diols, glycerol, and aminoalcohols, which are phosphorylated at the primary hydroxy-group, and inosine phosphorylated at the 5′-position Enzymatic regioselective phosphorylation of glycerol to racemic glycerol-1-phosphate with inorganic monophosphate has been achieved in a very large yield, first in 1991 using immobilized alkaline phosphatase [52] and recently with phytase. [59] Various substrates and suitable phosphoryl-donors were evaluated towards the synthesis of phosphate monoesters using alkaline phosphatases PhoK from Sphingomonas sp. [60] As the preparative resolution of racemic mixtures of phosphorylated metabolites requires substantial work, it is desirable to have direct routes to enantiomerically pure products.…”

“…Enzymatic regioselective phosphorylation of glycerol to racemic glycerol‐1‐phosphate with inorganic monophosphate has been achieved in a very large yield, first in 1991 using immobilized alkaline phosphatase [ 52 ] and recently with phytase. [ 59 ] Various substrates and suitable phosphoryl‐donors were evaluated towards the synthesis of phosphate monoesters using alkaline phosphatases PhoK from Sphingomonas sp. [ 60 ]…”

Enzymatic Production of Ascorbic Acid-2-Phosphate by Engineered Pseudomonas aeruginosa Acid Phosphatase

“…There has been increasing interest in AsA-2P production via enzymatic conversion using acid phosphatase (APase; EC 3.1.3.2) or whole-cell biocatalysts containing APase. − Brevundimonas diminuta was isolated from soil samples to produce 27.5 g/L AsA-2P with a 19.7% conversion rate after optimizing the cell-growth and transformation conditions . Additionally, Pseudomonas azotocolligans selected from 715 strains of microorganisms demonstrated the production of 31.6 g/L AsA-2P with a conversion rate of 27.6% .…”