Evaluation of Natural and Synthetic Phosphate Donors for the Improved Enzymatic Synthesis of Phosphate Monoesters

Tasnádi, Gábor; Jud, Wolfgang; Hall, Mélanie; Baldenius, Kai; Ditrich, K.; Faber, Kurt

doi:10.1002/adsc.201800306

Cited by 17 publications

(27 citation statements)

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“…High concentrationso fg lycerol[ pure glycerol( 1a)a nd crude glycerol( 1b)] and PP i as the P-donor were employed in overnight reactions at 30 8Ci nw ater.I mportantly,n op Ha djustmentw as necessary as the use of ah igh concentration of PP i (used as the disodium pyrophosphate salt, Na 2 H 2 P 2 O 7 )r esulted in a( buffered) acidic pH of approximately 3.5, av alue that was beneficial in ap revious study to reduce unwanted product hydrolysis in transphosphorylation reactions. [27] Pure 1a andc rude 1b (obtained from the base-catalyzed transesterificationo ffats with methanol) were well tolerated as substrates by most enzymes in up to 80 wt %b y using 5wt% PP i (Figures S1 and S2). The best results were obtained by using PhoC-Mm with 60 wt % 1a over 24 h, which led to a1 00 %P P i consumption,a 46 gL À1 product titer,a nd a0 .88 product/P i ratio (Figure S1), which corresponds to 94 %t ransphosphorylation efficiency.…”

Section: Resultsmentioning

confidence: 99%

“…High concentrations of glycerol [pure glycerol ( 1 a ) and crude glycerol ( 1 b )] and PP i as the P‐donor were employed in overnight reactions at 30 °C in water. Importantly, no pH adjustment was necessary as the use of a high concentration of PP i (used as the disodium pyrophosphate salt, Na 2 H 2 P 2 O 7 ) resulted in a (buffered) acidic pH of approximately 3.5, a value that was beneficial in a previous study to reduce unwanted product hydrolysis in transphosphorylation reactions …”

Section: Resultsmentioning

confidence: 99%

“…Acommon drawback of phosphatase-catalyzed transphosphorylation reactions lies in the undesired enzyme-catalyzed hydrolysis of the newly formed products.I na ddition to the use of high loadings of both alcohols ubstrate and P-donor, [19][20][21][22] varioust echniques have been developed to minimize product hydrolysis, such as continuous-flows ynthesis, [23,24] enzymee ngineering, [25,26] and reaction engineering. [27] They could enable the mild and selective enzymatic synthesis of phosphate monoesters of functionalized alcohols and sugars.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast, phytases could utilize 20 wt % NaH 2 PO 4 as the monophosphate salt for the direct condensation in 64 wt % glycerol to furnish 104 g L −1 product. In comparison, calf intestine alkaline phosphatase, which also delivers approximately 10 % of glycerol‐2‐phosphate in the phosphorylation of glycerol, could deliver 71 g L −1 product at best, whereas the product titer with nonspecific acid phosphatases reached only approximately 17 g L −1 from pyrophosphate under standard conditions . Importantly, several enzymes could operate efficiently in crude glycerol obtained from biodiesel production.…”

Section: Discussionmentioning

confidence: 99%

“…In comparison, calf intestine alkaline phosphatase, which also delivers approximately 10 %o fg lycerol-2-phosphate in the phosphorylation of glycerol, [17] couldd eliver 71 gL À1 product at best, [18] whereas the product titer with nonspecific acid phosphatases reachedo nly approximately 17 gL À1 from pyrophosphate under standard conditions. [26,27] Importantly,s everal enzymes could operate efficiently in crude glycerolo btained from biodiesel production. Moreover,p hytaseccw as able to work under neat conditions, which enabled the development of a preparative-scale synthesis on a1 00 mL scale and the isolation of rac-glycerol-1-phosphate by fractional crystallization as a magnesium salt in 26 %yield (16.6 g).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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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 %).

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

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

et al. 2020

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

Wohlgemuth

2020

Biotechnology Journal

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Biocatalytic phosphorylation reactions provide several benefits, such as more direct, milder, more selective, and shorter access routes to phosphorylated products. Favorable characteristics of biocatalytic methodologies represent advantages for in vitro as well as for in vivo phosphorylation reactions, leading to important advances in the science of synthesis towards bioactive phosphorylated compounds in various areas. The scope of this review covers key advances of biocatalytic phosphorylation reactions over the last two decades, for biocatalytic syntheses in vitro and for biotransformations in vivo (in humans). From the origins of probiotic life to in vitro synthetic applications and in vivo formation of bioactive pharmaceuticals, the common purpose is to outline the importance, relevance, and underlying connections of biocatalytic phosphorylations of small molecules. Asymmetric phosphorylations attracting increased attention are highlighted. Phosphohydrolases, phosphotransferases, phosphorylases, phosphomutases, and other enzymes involved in phosphorus chemistry provide powerful toolboxes for resource-efficient and selective in vitro biocatalytic syntheses of phosphorylated metabolites, chiral building blocks, pharmaceuticals as well as in vivo enzymatic formation of biologically active forms of pharmaceuticals. Nature's large diversity of phosphoryl-group-transferring enzymes, advanced enzyme and reaction engineering toolboxes make biocatalytic asymmetric phosphorylations using enzymes a powerful and privileged phosphorylation methodology. K E Y W O R D S chiral building blocks, enzymatic phosphorylation, kinases, metabolites, pharmaceuticals, phosphatases, phosphomutases, phosphoryl-donor, phosphorylases, phosphotransferases 1 INTRODUCTION Biocatalysis and biotransformations involving the element phosphorus are fundamental bioprocesses. The exciting history of the element Abbreviations: API, active pharmaceutical ingredient; FDA, U.S. Food and Drug Administration; P(III)-Donor, donor group containing phosphorus in its oxidation state +3; P(V)-Donor, donor group containing phosphorus in its highest oxidation state +5 phosphorus has been linked to life and death on our planet from its discovery from human urine in 1669 by alchemist Hennig Brand. Many phosphorus bonds to build phosphorus-containing molecules have been synthesized by biocatalytic phosphorylations, either by nature or anthropogenic. [1,2] As many phosphorus-bearing species are structurally stable and functionally reactive, they are also of interest as signals of life and have been detected recently in space. [3,4] The discovery of phosphorus-bearing species in space is of much interest

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Biocatalytic Production of Bioactive Dipeptides

Bordewick¹,

Berger²

2023

Lebensmittelchemie

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Dipeptides are a promising substance class with a large variety of bioactivities. N‐terminal ar‐ ginyl dipeptides (Arg‐X) have been shown to exhibit both salt‐taste enhancing (Arg‐Ser among others) and antihypertensive properties (Arg‐Phe). A novel biocatalytic strategy for the specific synthesis of these dipeptides is the application of L‐amino acid ligases like RizA from B. subtilis, which enables production of Arg‐X dipeptides directly from their amino acids. However, challenges for an industrial application are the dependence on the expensive cofactor ATP (adenosine triphosphate), low product yields, formation of the undesired side product Arg Arg and the potentially high enzyme costs.In order to reduce the need for ATP, a strategy for ATP regeneration from acetyl phosphate was established using acetate kinase from E. coli (AckA). Up to 5.9 g/L Arg‐Ser were produced, corresponding to an ATP efficiency of 23 g Arg‐Ser per gram ATP (without regeneration max. 0.5 g Arg‐Ser/g ATP). Next, variants of RizA were created to alter the substrate specificity. A total of 21 RizA variants were created by site‐directed mutagenesis of eight positions in the substrate binding pocket. Additional 14 variants were created by combination of different mutations. Variants with improved activity and specificity were identified for all five examined amino acid combinations (Arg + Arg, Asp, Ser, Ala, Phe). The largest change was observed for K83F_S156A, which improved the product formation of Arg‐Phe six‐fold and improved specificity more than tenfold. Finally, co immobilization of RizA and selected variants with AckA was performed to enable reusability of this biocatalytic system. Immobilisates of T81F_A158S produced up to 3.0 g/L Arg Ser and immobilisates of K83F_S156A produced up to 3.8 g/L Arg‐Phe and could both be reused several times.This thesis represents the first extensive scientific investigation into establishing ATP regeneration for an L‐amino acid ligase and co‐immobilization of such a biocatalytic system, and substantially expanded the knowledge about engineering the substrate specificity of L amino acid ligases.

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Evaluation of Natural and Synthetic Phosphate Donors for the Improved Enzymatic Synthesis of Phosphate Monoesters

Cited by 17 publications

References 50 publications

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

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

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

Biocatalytic Production of Bioactive Dipeptides

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