Comparative characterisation of thiamin diphosphate-dependent decarboxylases

Several 2-keto acid decarboxylases catalyse an acyloin condensation-like carboligase reaction beside their physiological decarboxylase activity. Although many data concerning stability and catalytic potential of these enzymes are available, a standard evaluation under similar reaction conditions is lacking. In this comprehensive survey we assemble already published data combined with new studies of three bacterial pyruvate decarboxylases, yeast pyruvate decarboxylase, benzoylformate decarboxylase from Pseudomonas putida (BFD) and the branched-chain 2-keto acid decarboxylase from Lactococcus lactis (KdcA). The obtained results proof that the optima for activity and stability are rather similar if comparable reaction conditions are used. Although the substrate ranges of the decarboxylase reaction of the various pyruvate decarboxylases are similar as well, they differ remarkably from those of BFD and KdcA. We further show that the range of acceptable donor aldehydes for the carboligase reaction of a respective enzyme can be reliably predicted from the substrate range of decarboxylase reaction.     

Carboligation Reactions Mediated by Benzoylformate Decarboxylase Immobilized on a Magnetic Solid Support

In this study, magnetic nanoparticles (Fe₃O₄, magnetite) with immobilized metal affinity ligands (MSS) were prepared and characterized by X‐ray diffraction (XRD), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), and vibrating‐sample magnetometer (VSM) methods for purification and immobilization of the histidine‐tagged recombinant benzoylformate decarboxylase (BFD). The MSS support was shown to be eligible for selective binding of HIS‐tagged BFD by SDS‐page analysis. Loading capacity of the MSS support was determined as 43.6 ± 1.1 mg/g. The regeneration ability for protein binding was also studied. An immobilized BFD was tested to catalyze benzoin condensation and representative cross acyloin reaction. Conversion and enantiomeric excess values were comparable with that of free enzyme catalyzed reactions. Chirality 25:415–421, 2013. © 2013 Wiley Periodicals, Inc.     

Propioin synthesis using thiamine diphosphate‐dependent enzymes

Benzaldehyde lyase (BAL, EC 4.1.2.38) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD, EC 4.1.1.7) from Pseudomonas putida are thiamine diphosphate‐dependent enzymes. These enzymes share a common tetrameric structure and catalyze various C? C‐bond forming and breaking reactions. Here we describe a detailed study of the asymmetric synthesis of propioin from propanal catalyzed by BAL or BFD in aqueous solution in a batch reactor. Both enzymes are deactivated in the presence of high concentration of propanal. Compared to BAL, BFD is more stable under reaction conditions as well as during storage. The kinetic studies showed a typical Michaelis‐Menten kinetic for BAL with a maximal specific reaction rate of 26.2 U/mg and an unusually high K_M of 415 mM, whereas the v/[S]‐plot for BFD is almost linear in the concentration range (100–1500 mM) investigated. Both enzymes produce propioin with opposite enantiomeric excess: BAL produced the (S)‐propioin (ee of 35%), whereas BFD yielded the (R)‐enantiomer (ee of 67%). © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Enzymatic production of (R)-phenylacetylcarbinol by pyruvate decarboxylase from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

Herein, we synthesized (R)-phenylacetylcarbinol (PAC), a pharmaceutical intermediate for ephedrine and pseudoephedrine, from benzaldehyde and pyruvate using a recombinant pyruvate decarboxylase (PDC) from Zymomonas mobilis. A whole cell reaction consisting of 30 mM benzaldehyde, 60 mM pyruvate, and a mutant PDC enzyme (PDC W329M; 1.6 mg DCW/mL) produced 12.4 mM (R)-PAC and less than 0.3 mM benzyl alchohol in 15 h at 20°C, outperforming the crude enzyme extract reaction (1.2 mM (R)-PAC) and minimizing formation of benzyl alchohol, the major by-product of S. cerevisiae whole cell reaction. These observations suggested that recombinant E. coli whole cell reactions are more efficient than crude enzyme extract or yeast-based reactions. We also demonstrated that the E. coli whole cell reaction performed effectively without expensive thiamin diphosphate cofactor. Finally, whole cell reaction (8 mg DCW/mL) was carried out with 200 mM benzaldehyde, 400 mM pyruvate in 10 mL of 500 mM phosphate buffer (pH 6.5), and 72 mM (R)-PAC was produced with 36% conversion for 15 h. © KSBB     

Continuous production of (R)-phenylacetylcarbinol in an enzyme-membrane reactor using a potent mutant of pyruvate decarboxylase from Zymomonas mobilis.

The optimization of a continuous enzymatic reaction yielding (R)-phenylacetylcarbinol (PAC), an intermediate of the L-ephedrine synthesis, is presented. We compare the suitability of three pyruvate decarboxylases (PDC), PDC from Saccharomyces cerevisiae, PDC from Zymomonas mobilis, and a potent mutant of the latter, PDCW392M, with respect to their application in the biotransformation using acetaldehyde and benzaldehyde as substrates. Among these, the mutant enzyme was the most active and most stable one. The reaction conditions of the carboligation reaction were investigated by determining initial rate velocities with varying substrate concentrations of both aldehydes. From the resulting data a kinetic model was inferred which fits the experimental data with sufficient reliability to deduce the optimal concentrations of both substrates for the enzymatic process. The results demonstrate that the carboligation is most efficiently performed using a continuous reaction system and feeding both aldehydes in equimolar concentration. Initial studies using a continuously operated enzyme-membrane reactor gave (R)-PAC with a space-time yield of 81 g L(-1). d(-1) using a substrate concentration of 50 mM of both aldehydes. The yield was easily increased by cascadation of enzyme-membrane reactors. The new strategy allows the synthesis of (R)-PAC from cheap substrates in an aqueous reaction system. It thereby overcomes the limitation of by-product formation that severely limits the current fermentative process.