Kinetics of Pyruvate Decarboxylase Deactivation by Benzaldehyde

Based on experimental data, a kinetic model for the deactivation of partially purified pyruvate decarboxylase (PDC) by benzaldehyde (0-200 mM) in MOPS buffer (2.5 M) has been developed. An initial lag period prior to deactivation was found to occur. With first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration, a reaction time deactivation constant of 2.64×10^-3 h^-1 and a benzaldehyde deactivation coefficient of 1.98×10^-4 mM^-1 h^-1 were determined for benzaldehyde concentrations up to 200 mM. The PDC deactivation kinetic equations established in this study are an essential component in an overall model being developed to describe the enzymatic biotransformation of benzaldehyde and pyruvate to produce the pharmaceutical intermediate (R)-phenylacetylcarbinol (R-PAC).     

A kinetic model for the deactivation of pyruvate decarboxylase (PDC) by benzaldehyde

To provide further understanding of the biotransformation of benzaldehyde to L-phenylacetyl carbinol (L-PAC), an intermediate in L-ephedrine production, a kinetic model has been developed for the deactivation of pyruvate decarboxylase (PDC) by benzaldehyde. The model confirms that deactivation is first order with respect to benzaldehyde concentration and exhibits a square root dependency on time. The model covers the range of benzaldehyde concentrations 100–300 mM, as it has been shown previously that 200 mM benzaldehyde can produce L-PAC concentrations up to 190 mM (28.6 g/L) using partially purified PDC from Candida utilis.     

Enzymatic (R)-phenylacetylcarbinol production in a benzaldehyde emulsion system with Candida utilis cells

Recent progress in enzymatic (R)-phenylacetylcarbinol (PAC) production has established the need for low cost and efficient biocatalyst preparation. Pyruvate decarboxylase (PDC) added in the form of Candida utilis cells showed higher stability towards benzaldehyde and temperature in comparison with partially purified preparations. In the presence of 50 mM benzaldehyde and at 4°C, a half-life of 228 h was estimated for PDC added as C. utilis cells, in comparison with 24 h for the partially purified preparation. Increasing the temperature from 4 to 21°C for PAC production with C. utilis cells resulted in similar final PAC levels of 39 and 43 g l⁻¹ (258 and 289 mM), respectively, from initial 300 mM benzaldehyde and 364 mM pyruvate. The overall volumetric productivity was enhanced 2.8-fold, which reflected the 60% shorter reaction time at the higher temperature. Enantiomeric excess values of 98 and 94% for R-PAC were obtained at 4 and 21°C, respectively, and benzyl alcohol (a potential by-product from benzaldehyde) was not formed.     

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     

Improved enzymatic two-phase biotransformation for (R)-phenylacetylcarbinol: Effect of dipropylene glycol and modes of pH control

An octanol/aqueous two-phase process for the enzymatic production of (R)-phenylacetylcarbinol (PAC) has been investigated further with regard to optimal pH control and replacement of 2.5?M MOPS buffer by a low cost solute. The specific rate of PAC production in the 2.5?M MOPS system controlled at pH?7 was 0.60?mg?U^?1?h^?1 (reaction completed at 34?h), a 1.6 times improvement over the same 2.5?M MOPS system without pH control (0.39?mg?U^?1?h^?1 at 49?h). An improved stability of PDC was evident at the end of biotransformation for the pH-controlled system with 84% residual carboligase activity, while 23% of enzyme activity remained in the absence of pH control. Lowering the MOPS concentration to 20?mM resulted in a lower benzaldehyde concentration in the aqueous phase with a major increase in the formation of by-product acetoin and three times decreased PAC production (0.21?mg?U^?1?h^?1). Biotransformation with 20?mM MOPS and 2.5?M DPG as inexpensive replacement of high MOPS concentrations provided similar aqueous phase benzaldehyde concentrations compared to 2.5?M MOPS and resulted in a comparable PAC concentration (92.1?g?L^?1 in the total reaction volume in 47?h) with modest formation of acetoin.     

Increased pyruvate efficiency in enzymatic production of (R)-phenylacetylcarbinol

Loss of substrate, pyruvate, a limitation for enzymatic batch production of (R)-phenylacetylcarbinol (PAC), resulted from two phenomena: temperature dependent non-enzymatic concentration decrease due to the cofactor Mg²⁺ and formation of by-products, acetaldehyde and acetoin, by pyruvate decarboxylase (PDC). In the absence of enzyme, pyruvate stabilization was achieved by lowering the Mg²⁺ concentration from 20 to 0.5 mM. With 0.5 mM Mg²⁺ Rhizopus javanicus and Candida utilis PDC produced similar levels of PAC (49 and 51 g l^–1, respectively) in 21 h at 6 °C; however C. utilis PDC formed less by-product from pyruvate and was more stable during biotransformation. The process enhancements regarding Mg²⁺ concentration and source of PDC resulted in an increase of molar yield (PAC/consumed pyruvate) from 59% (R. javanicus PDC, 20 mM Mg²⁺) to 74% (R. javanicus PDC, 0.5 mM Mg²⁺) to 89% (C. utilis PDC, 0.5 mM Mg²⁺).     

Role of pyruvate in enhancing pyruvate decarboxylase stability towards benzaldehyde

Biotransformation of benzaldehyde and pyruvate into (R)-phenylacetylcarbinol (PAC) catalysed by Candida utilis pyruvate decarboxylase (PDC) at low buffer concentration (20 mM MOPS) was enhanced by maintenance of neutral pH through acetic acid addition. PDC was very stable in this buffer (half-life 138 h at 6 degrees C), however a benzaldehyde emulsion (400 mM) caused rapid deactivation. The inclusion of 2M glycerol did not protect PDC from inactivation by benzaldehyde but initial rates were increased by 50% and the final PAC level was enhanced from 40 to 51 g l(-1). Low levels of by-products acetaldehyde (0.1-0.15 g l(-1)) and acetoin (1.1-1.3 g l(-1)) were formed in both the presence and absence of 2 M glycerol. Interestingly PDC was more stable towards benzaldehyde when pyruvate was present: no activity was lost during the first hour of biotransformation (2 M glycerol, benzaldehyde concentration decreased from 400 to 345 mM, pyruvate from 480 to 420 mM) but PDC was completely inactivated in less than 30 min when exposed to the same concentrations of benzaldehyde in the absence of pyruvate. Thus the enzyme in catalytic action was more stable than the resting enzyme.     

Zymobacter palmae pyruvate decarboxylase production process development: Cloning in Escherichia coli,fed‐batch culture and purification

Pyruvate decarboxylase (PDC) is responsible for the decarboxylation of pyruvate, producing acetaldehyde and carbon dioxide and is of high interest for industrial applications. PDC is a very powerful tool in the enzymatic synthesis of chiral amines by combining it with transaminases when alanine is used as amine donor. However, one of the main drawback that hampers its use in biocatalysis is its production and the downstream processing on scale. In this paper, a production process of PDC from Zymobacter palmae has been developed. The enzyme has been cloned and overexpressed in Escherichia coli. It is presented, for the first time, the evaluation of the production of recombinant PDC in a bench‐scale bioreactor, applying a substrate‐limiting fed‐batch strategy which led to a volumetric productivity and a final PDC specific activity of 6942 U L^?1h^?1 and 3677 U gDCW^?1 (dry cell weight). Finally, PDC was purified in fast protein liquid chromatography equipment by ion exchange chromatography. The developed purification process resulted in 100% purification yield and a purification factor of 3.8.     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l⁻¹ (PDC activity 2.5 U ml⁻¹), PAC levels of 103 g l⁻¹ in the octanol phase and 12.8 g l⁻¹ in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U⁻¹ h⁻¹) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l⁻¹ h⁻¹) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g⁻¹).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.     

Competitive substrate inhibition of amyloglucosidase from Rhizopus sp. by vanillin and curcumin

Kinetic studies of two glucosylation reactions catalyzed by an amyloglucosidase from Rhizopus sp. leading to the synthesis of vanillin-α/β-D-glucoside from D-glucose and vanillin and curcumin-bis-α-D-glucoside from D-glucose and curcumin were investigated in detail. Initial reaction rates were determined from kinetic runs involving different concentrations of D-glucose and vanillin (5?mM to 0.1?M) or D-glucose and curcumin (5?mM to 0.1?M). Graphical double reciprocal plots showed that the kinetics of the two enzyme catalyzed reactions exhibited Ping-Pong Bi-Bi mechanism where competitive substrate inhibition by vanillin/curcumin led to dead-end amyloglucosidase–vanillin/curcumin complexes at higher concentrations of vanillin/curcumin. An attempt to obtain the best fit of this kinetic model through computer simulation yielded in good approximation, the values of four important kinetic parameters, vanillin-α/β-D-glucoside: k_cat=35.0±3.2 10^?5M?h^?1·mg, K_i=10.5±1.1?mM, K_mD-glucose=60.0±6.2?mM, K_mvanillin=50.0±4.8?mM; curcumin-bis-α-D-glucoside: k_cat=6.07±0.58 10^?5M?h^?1·mg, K_i=3.0±0.28?mM, K_mD-glucose=10.0±0.9?mM, K_mcurcumin=4.6±0.5?mM.     

Increase in Silk Production by the Silkworm,Bombyx morì L., due to Oral Administration of a Juvenile Hormone Analog

(R)-1,3-butanediol ((R)-1,3-BD) is an important substrate for the synthesis of industrial chemicals. Despite its large demand, a bioprocess for the efficient production of 1,3-BD from renewable resources has not been developed. We previously reported the construction of recombinant Escherichia coli that could efficiently produce (R)-1,3-BD from glucose. In this study, the fermentation conditions were optimized to further improve 1,3-BD production by the recombinant strain. A batch fermentation was performed with an optimized overall oxygen transfer coefficient (82.3?h^?1) and pH (5.5); the 1,3-BD concentration reached 98.5?mM after 36?h with high-yield (0.444?mol (mol glucose)^?1) and a high maximum production rate (3.63?mM?h^?1). In addition, a fed-batch fermentation enabled the recombinant strain to produce 174.8?mM 1,3-BD after 96?h cultivation with a yield of 0.372?mol (mol glucose)^?1, a maximum production rate of 3.90?mM?h^?1, and a 98.6% enantiomeric excess (% ee) of (R)-1,3-BD.     

Continuous biodegradation of parathion by immobilized Sphingomonas sp. in magnetically fixed-bed bioreactors and evaluation of the enzyme stability of immobilized bacteria

Magnetically-modified Sphingomonas sp. was prepared using covalent binding of magnetic nanoparticles on to the cell surface. The magnetic modified bacteria were immobilized in the fixed-bed bioreactors (FBR) by internal and external magnetic fields for the biodetoxification of a model organophosphate, parathion: 93 % of substrate (50 mg parathion/l) was hydrolyzed at 0.5 ml/min in internal magnetic field fixed-bed bioreactor. The deactivation rate constants (at 1 ml/min) were 0.97 × 10^?3, 1.24 × 10^?3 and 4.17 × 10^?3 h^?1 for immobilized bacteria in external and internal magnetic field fixed-bed bioreactor and FBR, respectively. The deactivation rate constant for immobilized magnetically modified bacteria in external magnetic field fixed-bed bioreactor (EMFFBR) was 77 % lower than that of immobilized cells by entrapping method on porous basalt beads in FBR at 1 ml/min. Immobilized magnetic modified bacteria exhibited maximum enzyme stability in EMFFBR.     

Production of L-phenylacetylcarbinol (L-PAC) from benzaldehyde using partially purified pyruvate decarboxylase (PDC)

Biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) as a key intermediate for L-ephedrine synthesis has been evaluated using pyruvate decarboxylase (PDC) partially purified from Candida utilis. PDC activity was enhanced by controlled fermentative metabolism and pulse feeding of glucose prior to the enzyme purification. With partially purified PDC, several enzymatic reactions occurred simultaneously and gave rise to by-products (acetaldehyde and acetoin) as well as L-PAC production. Optimal reaction conditions were determined for temperature, pH, addition of ethanol, PDC activity, benzaldehyde, and pyruvate:benzaldehyde ratio to maximize L-PAC, and minimize by-products. The highest L-PAC concentration of 28.6 g/L (190.6 mM) was achieved at 7 U/mL PDC activity and 200 mM benzaldehyde with 2.0 molar ratio of pyruvate to benzaldehyde in 40 mM potassium phosphate buffer (pH 7.0) containing 2.0 M ethanol at 4 degrees C. (c) 1996 John Wiley & Sons, Inc.     

Bacterial dynamics in two high-arctic lakes

The heterotrophic planktonic bacteria in two high-arctic lakes were studied by direct microscope count and the enzymatic uptake of ¹⁴C labelled glucose which generally conformed to Michaelis-Menten kinetics. Bacterial numbers and activity in oligo-trophic Char Lake ranged from 0.1 to 2.0×10^?3 bacteria/l and a maximum uptake velocity (V_max) of 1.8 × 10^?3μg glucose l^? h^?1. Nearby Meretta Lake received waste water from the Department of Transport Base at Resolute and this eutrophication was reflected in higher bacterial numbers of 2-80 × 10⁸/1 and Kmax of 0.1 × 10^?1-7.5 × 10^?1 fig glucose l^?1 h^?1 The Kmax per cell in Char Lake was 3 × 10^?11μg glucose l^?1 h^?1 and changed little between the period of solid ice cover in May and ice-free conditions in August. Bacterial cycles could not be related to phytoplankton cycles in either lake. Comparison of kinetic data from several lakes suggests a relationship between the bacterial uptake rate of glucose and phytoplankton production. Both bacterial numbers and activity in Char Lake may be very close to the minima to be expected in undisturbed freshwater environments.     

Enzymatic synthesis of new aromatic and aliphatic esters of flavonoids using Candida antarctica lipase as biocatalyst

Enzymatic acylation of rutin and esculin with aromatic, aliphatic and aryl-aliphatic acids using Candida antarctica lipase in tert amyl alcohol as solvent was investigated under low water content. Whatever the acyl donor used, the conversion yields and initial rates for esculin were higher than for rutin. For a given flavonoid, the performance of these reactions depended on the acyl donor structures. For aliphatic acids, conversion yields and initial rates of both flavonoids were respectively in the ranges of 68–90% and of 9.5×10^?2–72×10^?2 mmol l^?1 h^?1. For aromatic acids, the reaction occurred only with the aryl subgroup (cinnamic, hydrocinnamic, 3,4-dihydroxyhydrocinnamic and 4-hydroxyphenyl acetic acids) and was drastically influenced by the presence of side chain and substitution patterns of the aromatic ring. Except for hydrocinnamic acid (75%, 23.4×10^?2 mmol l^?1 h^?1), with these acids the conversion yields and initial rates were lower and in the range of 10–45% and of 0.7×10^?2 to 12.1×10^?2 mmol l^?1 h^?1. Unsaturation of the side chain of the hydrocinnamic acid decreased the esculin conversion rate from 75 to 13% and initial rate from 23.4 to 1.76×10^?2 mmol l^?1 h^?1. The presence of hydroxyl or nitro-groups on the aromatic ring of the aryl aliphatic acid also reduced conversion yields and initial rates. Even without a spacer, the non-phenolic ring acid (quinic acid) was reactive and lead to conversion yields of about 20 and 23% respectively for rutin and esculin.     

Kinetic analysis and modelling of enzymatic (R)-phenylacetylcarbinol batch biotransformation process

Initial rate and biotransformation studies were applied to refine and validate a mathematical model for enzymatic (R)-phenylacetylcarbinol (PAC) production from pyruvate and benzaldehyde using Candida utilis pyruvate decarboxylase (PDC). The rate of PAC formation was directly proportional to the enzyme activity level up to 5.0 U ml-1 carboligase. Michaelis-Menten kinetics were determined for the effect of pyruvate concentration on the reaction rate. The effect of benzaldehyde followed the sigmoidal shape of the Monod-Wyman-Changeux (MWC) model. The biotransformation model, which also included a term for PDC inactivation by benzaldehyde, was used to determine the overall rate constants for the formation of PAC, acetaldehyde, and acetoin. These values were determined from data for three batch biotransformations performed over a range of initial concentrations (viz. 50-150 mM benzaldehyde, 60-180 mM pyruvate, 1.1-3.4 U ml-1 enzyme activity). The finalized model was then used to predict a batch biotransformation profile at 120/100 mM initial pyruvate/benzaldehyde (initial enzyme activity 3.0 U ml-1). The simulated kinetics gave acceptable fitting (R2 = 0.9963) to the time courses of these latter experimental data for substrates pyruvate and benzaldehyde, product PAC, by-products acetaldehyde and acetoin, as well as enzyme activity level.     

Screening of yeasts for cell-free production of (R)-phenylacetylcarbinol

105 yeast strains from 10 genera and 40 species were evaluated for cell-free production of (R)-phenylacetylcarbinol (PAC), the chiral precursor in the manufacture of the pharmaceuticals ephedrine and pseudoephedrine. Carboligase activity of pyruvate decarboxylase (PDC), forming PAC from benzaldehyde and pyruvate, was found in extracts of 98 strains. PAC was not formed from benzaldehyde and acetaldehyde, an activity of bacterial PDCs from Zymomonas mobilis and Zymobacter palmae. Two interesting groups of candidates were identified in the yeast screening: carboligase activities of Schizosaccharomyces pombe PDCs were very low but showed best resistance to pre-incubation with acetaldehyde and benzaldehyde; and highest carboligase activities combined with medium resistance were found in strains of Candida utilis, C. tropicalis and C. albicans.     

Growth kinetics of top and bottom cultures of Saccharomyces spp. in a chemostat using sugarbeet molasses

Growth kinetics were evaluated for three yeast strains of the genus Saccharomyces. Two topfloating strains, SF 115 and SF 116 and one flocculant yeast SF 104 were analyzed in pure and mixed cultures in 1-liter continuous fermentation experiments in a chemostat. Growth was monitored for 72 h at 30°C in a medium containing sugarbeet molasses and 1.0 g/liter each of NH₄H₂PO₄ and urea. SF 115 and SF 116 were found to have lower μ_max values of 0.290 and 0.296 h^?1, respectively, than SF 104, which had a μ_max of 0.364 h^?1. The two top-floating yeasts (SF 115 and SF 116) demonstrated greater affinity for the substrate and utilized substrates at a greater rate. They have K₈ values of 4.03 × 10^?3 M and 3.798 × 10^?3 M, respectively, compared to 9.06 × 10^?3 M for SF 104. A mixed culture of SF 116 and SF and SF 104 was found to have a μ_max of 0.426 h^?1 with a K_s of 6.924 × 10^?3 M. SF 115 grown in mixed culture with SF 104 exhibited a μ_max of 0.473 h^?1 with a K_s of 7.975 × 10^?3 M. In both cases, the SF 104 was the dominant microbe in mixed culture systems.     

Translational and rotational diffusion of tobacco mosaic virus from polarized and depolarized light scattering

The translational and rotational dynamics of tobacco mosaic virus in sodium phosphate buffer (pH =7.5) solutions has been investigated by polarized and depolarized light scattering Rayleigh linewidth studies. For concentrations ranging from 1.75 × 10^?4 g ml^?1 to 0.25 × 10^?4 g ml^?1 the translational diffusion coefficient (D_T) has been found to be slightly concentration dependent and extrapolation to zero concentration gives D₀^20°C = 0.34 ± 0.01 × 10^?7 cm²S^?1. A full analysis of the polarized spectra obtained at high and low scattering angles and the depolarized spectra at near zero scattering angles has enabled these techniques to be compared and the rotational diffusion constant D_R to be determined. At a solution concentration of 1.75 × 10^?4 g ml^?1 a mean value is found to be D_R^20°C = 350 ± 30s^?1. These values of D_T and D_R are in approximate agreement with calculations based on models of the tobacco mosaic virus molecule as a cylindrical rod.     

Glutathione biosynthesis in murine L5178Y lymphoma cells

The pyruvate dehydrogenase complex from pea leaf mitochondria was rapidly deactivated in the presence of 50 to 200 μm ATP. The deactivation of the complex requires Mg²⁺ as shown by EDTA inhibition of deactivation. Deactivation was inhibited by 0.1 to 1 mm pyruvate or dichloroacetate. Activation required 10 mM Mg²⁺ or Mn²⁺ but Ca²⁺ and K⁺ had no effect. Activation was inhibited by the phosphatase inhibitor, F^?. Autoradiograms of nondissociating electrophoresis gel, crossed immunoelectrophoresis gels, and dissociating sodium dodecyl sulfate electrophoresis gels of the complex showed that one protein is labeled. Labeling of this protein is prevented by Mg²⁺, pyruvate, and dichloroacetate. The pyruvate dehydrogenase complex was isolated in a partially deactivated state and reactivation required exogenous Mg²⁺ and was inhibited by F^?. These results are taken as conclusive evidence that the pyruvate dehydrogenase complex in pea leaf mitochondria undergoes interconversion between deactivated and activated states by covalent modification (phosphorylation-dephosphorylation) catalyzed by a kinase and phosphatase. Isolation of the complex in a partially deactivated (phosphorylated) state suggests a physiologically significant role for this regulatory mechanism.