Browsing by Author "Stolz, Andreas"

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Open Access
Chemoenzymatic enantioselective synthesis of phenylglycine and phenylglycine amide by direct coupling of the Strecker synthesis with a nitrilase reaction
(2022) Eppinger, Erik; Gröning, Janosch Alexander David; Stolz, Andreas
The conversion of rac-phenylglycinonitrile by different variants of the nitrilase from Pseudomonas fluorescens EBC191 (EC 3.5.5.1) was studied and the amounts and chiral composition of the formed phenylglycine and phenylglycine amide compared. Muteins that converted rac-phenylglycinonitrile to extraordinarily high amounts of phenylglycine or phenylglycine amide were tested for the chemoenzymatic enantioselective one-pot synthesis of (R)- and (S)-phenylglycine and (R)- and (S)-phenylglycine amide. The chemoenzymatic synthesis combined the initial step in the traditional chemical Strecker synthesis which results in the formation of rac-phenylglycinonitrile from benzaldehyde, cyanide, and ammonia with the enzymatic conversion of the formed nitrile by the nitrilase variants. The aminonitrile synthesis was optimized in order to obtain conditions which allowed under mildly alkaline conditions (pH 9.5) maximal yields of phenylglycinonitrile and the in-situ racemization of the compound. The racemic phenylglycinonitrile was directly converted under the alkaline conditions without any interposed purification step by cells of Escherichia coli overexpressing recombinant nitrilase variants. The application of a mutant of E. coli defect in a (S)-phenylglycine amide hydrolysing peptidase (E. coli JM109ΔpepA) expressing a highly reaction- and (R)-specific nitrilase variant allowed the synthesis of (R)-phenylglycine with ee-values ≥ 95% in yields up to 81% in relation to the initially added benzaldehyde. These yields indicated a dynamic kinetic resolution which involved the racemization of (S)- to (R)-phenylglycinonitrile under the used alkaline conditions with the concurrent hydrolysis of (R)-phenylglycinonitrile to (R)-phenylglycine. The addition of resting cells of E. coli JM109ΔpepA synthesizing an amide forming nitrilase variant to the final product of the Strecker synthesis and/or using E. coli strains with an intact aminopeptidase gene resulted in the preferred formation of (S)-phenylglycine amide, (R)-phenylglycine amide or (S)-phenylglycine.
Open Access
Enantioselective hydrolysis of O-acetylmandelonitrile to O-acetylmandelic acid by bacterial nitrilases
(1992) Layh, Norman; Stolz, Andreas; Förster, Siegfried; Effenberger, Franz; Knackmuss, Hans-Joachim
Bacteria were enriched from soil samples, using benzylcyanide, α-methyl-, α-ethyl- or α-methoxybenzyl-cyanide as the sole source of nitrogen. All isolated strains belonged to the genus Pseudomonas. Resting cells of the isolates hydrolysed O-acetylmandelonitrile to O-acetylmandelic acid, O-acetylmandelic acid amide and mandelic acid. From racemic O-acetylmandelonitrile all isolates preferentially formed R(–)-acetylmandelic acid ( = d-acetylmandelic acid). The enantioselective hydrolysis of O-acetylmandelonitrile could also be demonstrated in vitro. Crude extracts did not hydrolyse O-acetylmandelic acid amide indicating an enantioselective nitrilase rather than a nitrile hydratase/amidase system.
Open Access
Enantioselective hydrolysis of racemic naproxen nitrile and naproxen amide to S-naproxen by new bacterial isolates
(1994) Layh, Norman; Stolz, Andreas; Böhme, Joachim; Effenberger, Franz; Knackmuss, Hans-Joachim
Bacteria were enriched from soil samples with succinate as a carbon source and racemic naproxen nitrile [2-(6-methoxy-2-naphthyl)propionitrile] as sole source of nitrogen. Since naproxen nitrile was only poorly soluble in water media amended with different water-immiscible organic phases were used for the enrichments. With pristane (2,6,10,14-tetramethylpentadecane) as the organic phase two bacterial strains were isolated (strain C3II and strain MP50) which were identified as rhodococci. Cells of both strains converted naproxen nitrile via naproxen amide to naproxen. From racemic naproxen nitrile Rhodococcus sp. C3II formed S-naproxen amide and subsequently S-naproxen. Racemic naproxen amide was hydrolysed to S-naproxen. Rhodococcus sp. MP50 converted racemic naproxen nitrile predominantly to R-naproxen amide and racemic naproxen amide to S-naproxen. With both strains racemic naproxen amide was converted to S-naproxen with an enantiomeric excess >99% at a conversion rate up to 80% of the theoretical value. In strain C3II the enzymes which hydrolysed naproxen nitrile and naproxen amide were present only at a low constitutive level. In contrast, in Rhodococcus sp. MP50 these activities were induced when grown in the presence of various nitriles.
Open Access
A method for 3D printing bio-cemented spatial structures using sand and urease active calcium carbonate powder
(2020) Nething, Christoph; Smirnova, Maya; Gröning, Janosch A. D.; Haase, Walter; Stolz, Andreas; Sobek, Werner
The substitution of Portland cement with microbially based bio-cement for the production of construction materials is an emerging sustainable technology. Bio-cemented building components such as bricks have been fabricated in molds, where bacteria-containing aggregates solidify when treated with a cementation solution. Thisrestricts component size due to the limitedfluid penetration depth and narrows options for component customization. The use of additive manufacturing technologies has the potential to overcome those limitations and toexpand the range of bio-cement applications. In the present work an automated process for the production ofspatial structures has been developed, in which sand and urease active calcium carbonate powder were selectively deposited within a print volumeand treatedwith a cementation solution.This method provided conditionsfor calcite precipitation in the powder-containing areas, whereas areas of pure sand served as removable supportstructure allowing improvedfluid exchange. The 3D printed structure was geometrically stable and had sharplydefined boundaries. Compressive strength tests on cylindricalspecimens showed thatthe used powder-sandmixwas suitable for the production of high-strength bio-cemented material. The present work demonstrates an application of bio-cement in an additive manufacturing process, that can potentially be used to produce resourceefficient sustainable building components.
Open Access
Synthesis of (R)-mandelic acid and (R)-mandelic acid amide by recombinant E. coli strains expressing a (R)-specific oxynitrilase and an arylacetonitrilase
(2020) Müller, Erik; Sosedov, Olga; Gröning, Janosch Alexander David; Stolz, Andreas
Chiral 2-hydroxycarboxylic acids and 2-hydroxycarboxamides are valuable synthons for the chemical industry. The biocatalytic syntheses of (R)-mandelic acid and (R)-mandelic acid amide by recombinant Escherichia coli clones were studied. Strains were constructed which simultaneously expressed a (R)-specific oxynitrilase (hydroxynitrile lyase) from the plant Arabidopsis thaliana together with the arylacetonitrilase from the bacterium Pseudomonas fluorescens EBC191. In addition, recombinant strains were constructed which expressed a previously described acid tolerant variant of the oxynitrilase and an amide forming variant of the nitrilase. The whole cell catalysts which simultaneously expressed the (R)-specific oxynitrilase and the wild-type nitrilase transformed in slightly acidic buffer systems benzaldehyde plus cyanide preferentially to (R)-mandelic acid with ee-values > 95%. The combination of the (R)-specific oxynitrilase with the amide forming nitrilase variant gave whole cell catalysts which converted at pH-values ≤ pH 5 benzaldehyde plus cyanide with a high degree of enantioselectivity (ee > 90%) to (R)-mandelic acid amide. The acid and the amide forming catalysts also converted chlorinated benzaldehydes with cyanide to chlorinated mandelic acid or chlorinated mandelic acid amides. Efficient systems for the biocatalytic production of (R)-2-hydroxycarboxylic acids and (R)-2-hydroxycarboxamides were generated.