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.     

Biphasic aqueous/organic biotransformation of acetaldehyde and benzaldehyde by Zymomonas mobilis pyruvate decarboxylase

Zymomonas mobilis pyruvate decarboxylase (PDC) transformed acetaldehyde and benzaldehyde into (R)-phenylacetylcarbinol (PAC), the precursor for the synthesis of ephedrine and pseudoephedrine. Organic solvents were screened for a biphasic biotransformation with the enzyme in an aqueous phase and the toxic substrates delivered through the organic phase. In the absence of substrates a second phase of 1-pentanol, hexadecane or MTBE (methyl tertiary-butyl ether) stabilized the PDC activity in comparison to a control without added solvent. Organic phase solvents for optimal PAC production had partitioning coefficient (log P) values between 0.8 and 2.8 (production of more than 8 mg PAC/ U PDC), however there was no correlation between enzyme stability and log P. Best PAC formation was observed with the eight tested alcohols, which in contrast to the other solvents allowed lower initial concentrations of toxic acetaldehyde (54-81 mM) in the aqueous phase. 1-pentanol, 1-hexanol, and isobutanol resulted in the highest specific PAC production of 11 mg PAC /U PDC. Without the addition of an organic phase, only 1.2 mg/U was formed.     

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.     

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.     

Comparative studies on enzyme preparations and role of cell components for (R)-phenylacetylcarbinol production in a two-phase biotransformation

Whole cell pyruvate decarboxylase (PDC) from Candida utilis enhanced the enzymatic production of (R)-phenylacetylcarbinol (PAC) in an aqueous/octanol biotransformation compared to the partially purified PDC especially for a lower range of initial activities (0.3-2.5 U/mL). With an initial activity of 1.1 U/mL and at a 1:1 phase volume ratio, whole cell PDC achieved a maximum specific PAC production of 42 mg/U (2.8 g/L/h) in comparison to 13 mg/U (0.9 g/L/h) for partially purified PDC. The enhanced performance of whole cell PDC was associated with high stability towards the substrate benzaldehyde. The strong PDC inactivation by benzaldehyde was minimal even when whole cells were broken as long as cell debris was not removed from the broken cells. Biotransformations with various cellular components added to partially purified PDC revealed that membrane components especially 2 mg/mL phosphatidylcholine enhanced PAC concentrations. The role of surfactants was further confirmed from the results with synthetic surfactant sodium bis(2-ethyl-1-hexyl)sulfosuccinate (AOT). It was apparent that the membrane components in whole cells were sufficient for optimal PAC production and no further surfactant addition is required for optimal performance.     

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.     

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.     

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”.     

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.     

Yeast pyruvate decarboxylases: variation in biocatalytic characteristics for (R)-phenylacetylcarbinol production

Based on previous studies, Candida utilis pyruvate decarboxylase (PDC) proved to be a stable and high productivity enzyme for the production (R)-phenylacetylcarbinol (PAC), a pharmaceutical precursor. However, a portion of the substrate pyruvate was lost to by-product formation. To identify a source of PDC which might overcome this problem, strains of four yeasts -- C. utilis, Candida tropicalis, Saccharomyces cerevisiae and Kluyveromyces marxianus -- were investigated for their PDC biocatalytic properties. Biotransformations were conducted with benzaldehyde and pyruvate as substrates and three experimental systems were employed (in the order of increasing benzaldehyde concentrations): (I) aqueous (soluble benzaldehyde), (II) aqueous/benzaldehyde emulsion, and (III) aqueous/octanol-benzaldehyde emulsion. Although C. utilis PDC resulted in the highest concentrations of PAC and was the most stable enzyme, C. tropicalis PDC was associated with the lowest acetoin formation. For example, in system (III) the ratio of PAC over acetoin was 35 g g(-1) for C. tropicalis PDC and 9.2 g g(-1) for C. utilis PDC. The study thereby opens up the potential to design a PDC with both high productivity and high yield characteristics.     

Kinetic evaluation of biotransformation of benzaldehyde to L-phenylacetylcarbinol by immobilized pyruvate decarboxylase from Candida utilis

Biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) as a key intermediate for L-ephedrine has been evaluated using immobilized pyruvate decarboxylase (PDC) from Candida utilis. PDC immobilized in spherical polyacrylamide beads was found to have a longer half-life compared with free enzyme. In a batch process, the immobilized PDC generally produced lower L-PAC than free enzyme at the same concentrations of substrates due to increased by-products acetaldehyde and acetoin and reduced benzaldehyde uptake. With immobilized PDC, L-PAC formation occurred at higher benzaldehyde concentrations (up to 300 mM) with the highest L-PAC concentration being 181 mM (27.1 g/L). For a continuous process, when 50 mM benzaldehyde and 100 mM sodium pyruvate were fed into a packed-bed reactor at 4 degrees C and pH 6.5, a productivity of 3.7 mM/h (0.56 g/L . h) L-PAC was obtained at an average concentration of 30 mM (4.5 g/L). The half-life of immobilized PDC reactor was 32 days. (c) 1996 John Wiley & Sons, Inc.     

Enzymatic (R)-phenylacetylcarbinol production in benzaldehyde emulsions

(R)-Phenylacetylcarbinol [(R)-PAC)] is the chiral precursor for the production of the pharmaceuticals ephedrine and pseudoephedrine. Reaction conditions were improved to achieve increased (R)-PAC levels in a simple batch biotransformation of benzaldehyde emulsions and pyruvate, using partially purified pyruvate decarboxylase (PDC) from the filamentous fungus Rhizopus javanicus NRRL 13161 as the catalyst. Lowering the temperature from 23 degrees C to 6 degrees C decreased initial rates but increased final (R)-PAC concentrations. Addition of ethanol, which increases benzaldehyde solubility, was not beneficial for (R)-PAC production. It was established that proton uptake during biotransformation increases the pH above 7 thereby limiting (R)-PAC production. For small-scale studies, biotransformations were buffered with 2-2.5 M MOPS (initial pH 6.5). High concentrations of MOPS as well as some alcohols and KCl stabilised PDC. A balance between PDC and substrate concentrations was determined with regards to ( R)-PAC production and yields on enzyme and substrates. R. javanicus PDC (7.4 U/ml) produced 50.6 g/l (337 mM) ( R)-PAC in 29 h at 6 degrees C with initial 400 mM benzaldehyde and 600 mM pyruvate. Molar yields on consumed benzaldehyde and pyruvate were 97% and 59%, respectively, with 17% pyruvate degraded and 24% converted into acetaldehyde and acetoin; 43% PDC activity remained, indicating reasonable enzyme stability at high substrate and product concentrations.     

Improved (<Emphasis Type="Italic">R</Emphasis>)-phenylacetylcarbinol production with <Emphasis Type="Italic">Candida utilis</Emphasis> pyruvate decarboxylase at decreased organic to aqueous phase volume ratios

The effect of decreasing the organic (octanol) to aqueous phase volume ratio was evaluated in a two-phase enzymatic process for (R)-phenylacetylcarbinol (PAC) production. Decreasing the ratio from 1:1 to 0.43:1 at 4°C increased PAC in the organic phase from 112 g/l to 183 g/l with a 10% improvement in overall productivity. Interestingly, the rate of enzyme (pyruvate decarboxylase) activity loss was unaffected by the reduced phase ratio over the reaction period (48 h). At 20°C and 0.43:1 phase ratio the organic phase PAC concentration increased to 212 g/l and the overall productivity increased by 30% although the PAC yield (based on pyruvate) declined by about 10% due to greater byproduct acetoin formation at the higher temperature. Product recovery in such a system is facilitated both by the higher PAC concentration and the reduced organic phase volume.     

Studies on the continuous production of (R)-(−)-phenylacetylcarbinol in an enzyme-membrane reactor

The optimization of a continuous enzymatic reaction yielding (R)-(−)-phenylacetylcarbinol ((R)-PAC), a key intermediate of the (1R,2S)-(−)-ephedrine synthesis, is presented. We compare the suitability of different mutants of the pyruvate decarboxylase (PDC) from Zymomonas mobilis with respect to their application in biotransformation using pyruvate or acetaldehyde and benzaldehyde as substrates, respectively. Starting from 90 mM pyruvate and 30 mM benzaldehyde, (R)-PAC was obtained with a space time yield of 27.4 g/(L·day) using purified PDCW392I in an enzyme-membrane reactor. Due to the high stability of the mutant enzymes PDCW392I and PDCW392M towards acetaldehyde, a continuous procedure using acetaldehyde instead of pyruvate was developed. The kinetic results of the enzymatic synthesis starting from acetaldehyde and benzaldehyde demonstrate that the carboligation to (R)-PAC is most efficiently performed using a continuous reaction system and feeding both aldehydes in equimolar concentration. Starting from an inlet concentration of 50 mM of both aldehydes, (R)-PAC was obtained with a space-time yield of 81 g/(L·day) using the mutant enzyme PDCW392M. The new reaction strategy allows the enzymatic synthesis of (R)-PAC from cheap substrates free of unwanted by-products with potent mutants of PDC from Z. mobilis in an aqueous reaction system.     

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⁺).     

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.     

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.     

Acetohydroxyacid synthase: a new enzyme for chiral synthesis of R-phenylacetylcarbinol

We have found that acetohydroxyacid synthase (AHAS) is an efficient catalyst for the enantiospecific (> or =98% enantiomeric excess) synthesis of (R)-phenylacetylcarbinol (R-PAC) from pyruvate and benzaldehyde, despite the fact that its normal physiological role is synthesis of (S)-acetohydroxyacids from pyruvate and a second ketoacid. (R)-phenylacetylcarbinol is the precursor of important drugs having alpha and beta adrenergic properties, such as L-ephedrine, pseudoephedrine, and norephedrin. It is currently produced by whole-cell fermentations, but the use of the isolated enzyme pyruvate decarboxylase (PDC) for this purpose is the subject of active research and development efforts. Some of the AHAS isozymes of Escherichia coli have important advantages compared to PDC, including negligible acetaldehyde formation and high conversion of substrates (both pyruvate and benzaldehyde) to PAC. Acetohydroxyacid synthase isozyme I is particularly efficient. The reaction is not limited to condensation of pyruvate with benzaldehyde and other aromatic aldehydes may be used.     

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