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Query: UMLS:C0033036 (APC)
 10,214 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The assumption that the pyruvate decarboxylase activity of Saccharomyces carlsbergensis is the main limiting factor determining the formation rate and the total amount of d(--)-l-hydroxy-l-phenyl-propanone (phenylacetylcarbinol, PAC) produced was not confirmed. An increase of about 30% of the total amount of the PAC produced was obtained when 8.5% sodium pyruvate was gradually added. The total PAC production is probably influenced both by the pyruvate decarboxylase activity and the pyruvate concentration in the cells, the latter being actually the determining rate-limiting factor.
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PMID:Effect of pyruvate decarboxylase activity and of pyruvate concentration on the production of 1-hydroxy-1-phenylpropanone in Saccharomyces carlsbergensis. 710 60

L-ephedrine is widely used in pharmaceutical preparations as a decongestant and anti-asthmatic compound. One of the key intermediates in its production is L-phenylacetylcarbinol (L-PAC) which can be obtained either from plants (Ephedra sp.), chemical synthesis involving resolution of a racemic mixture, or by biotransformation of benzaldehyde using various yeasts. In the present review, recent significant improvements in the microbial biotransformation are assessed for both fed-batch and continuous processes using free and immobilised yeasts. From previous fed-batch culture data, maximal levels of L-PAC of 10-12 gl-1 were reported with yields of 55-60% theoretical based on benzaldehyde. However, recently concentrations of more than 22 gl-1 have been obtained using a wild-type strain of Candida utilis. This has been achieved through optimal control of yeast metabolism (via microprocessor control of the respiratory quotient, RQ) in order to enhance substrate pyruvate production and induce pyruvate decarboxylase (PDC) activity. Processes involving purified PDC have also been evaluated and it has been demonstrated that L-PAC levels up to 28 gl-1 can be obtained with yields of 90-95% theoretical based on the benzaldehyde added. In the review the advantages and disadvantages of the various strategies for the microbial and enzymatic production of L-PAC are compared. In view of the increasing interest in microbial biotransformations, L-PAC production provides an interesting example of enhancement through on-line control of a process involving both toxic substrate (benzaldehyde) and end-product (L-PAC, benzyl alcohol) inhibition.
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PMID:Biotransformation for L-ephedrine production. 893 58

Production of L-phenylacetylcarbinol (L-PAC) through biotransformation of benzaldehyde by free and immobilized cells of the yeast Saccharomyces cerevisiae has been attempted. L-PAC production was found to be maximum (0.4 microliter/ml) when anaerobically grown free cells were used as biocatalyst during aerobic biotransformation for two hours with magnetically stirred bioreactor. Growth under oxygen limited conditions led to accumulation of higher amount of pyruvate decarboxylase enzyme and co-substrate, pyruvate, resulting in higher L-PAC formation. L-PAC yield was low when biotransformations were carried out anaerobically either for aerobically or anaerobically grown free cells. Free cells were found to be more efficient biocatalyst for L-PAC production, as compared with the immobilized cells, with the investigated benzaldehyde concentration (0.3% v/v) and cell density (17.5% w/v). The study has explored and indicated the possibility of optimizing the yield of L-PAC by growing the yeast cells under oxygen limited condition for suitable aerobic mode of benzaldehyde biotransformation.
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PMID:Production of L-phenylacetylcarbinol by free and immobilized yeast cells. 947 65

The homotetrameric pyruvate decarboxylase (PDC) from Zymomonas mobilis requires the cofactors thiamin diphosphate and Mg2+ for catalytic activity. We have investigated the role of various amino acid residues in the direct environment of the active site. The role of residue E473 in the catalytic activity and stability of the enzyme was probed by several mutations. All mutant enzymes were either inactive or failed to give any recombinant protein. The close interaction of E473 and N482, which can be deduced from the X-ray structure, has been probed by mutagenesis of N482 to D. This mutation has a significant influence especially on the carboligation reaction of PDC, whereas the binding of the cofactors and the thermostability were not affected. These data suggest a specific interaction of N482 and EA73 which is essential for coordinating the second aldehyde molecule during carboligation. Three hydrophobic residues (L112, I472 and I476) in the vicinity of the active centre have been investigated with respect to their potential influence on the transition states during catalysis. In contrast to L112, I472 and I476 influence the decarboxylation and carboligation reactions. The enlarged substrate-binding site of PDCI472A allows the decarboxylation of longer aliphatic 2-keto acids (C4-C6) as well as aromatic 2-keto acids besides pyruvate. Carboligations using PDCI472A as a catalyst yielded 2-hydroxypropiophenone, benzoin and phenylacetylcarbinol. The enantioselectivity of PAC formation is impaired by mutations of both I472 and I476. The stereochemistry is most significantly affected with the mutant enzyme PDCI476E, which catalyses predominantly the synthesis of (S)-phenylacetylcarbinol.
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PMID:Active site mutants of pyruvate decarboxylase from Zymomonas mobilis--a site-directed mutagenesis study of L112, I472, I476, E473, and N482. 983 41

L-Phenylacetylcarbinol (L-PAC) is the precursor for L-ephedrine and D-pseudoephedrine, alkaloids possessing alpha- and beta-adrenergic activity. The most commonly used method for production of L-PAC is a biological method whereby the enzyme pyruvate decarboxylase (PDC) decarboxylates pyruvate and then condenses the product with added benzaldehyde. The process may be undertaken by either whole cells or purified PDC. If whole cells are used, the biomass may be grown and allowed to synthesize endogenous pyruvate, or the cells may be used as a catalyst only, with both pyruvate and benzaldehyde being added. Several yeast species have been investigated with regard to L-PAC-producing potential; the most commonly used organisms are strains of Saccharomyces cerevisiae and Candida utilis. It was found that initial high production rates did not necessarily result in the highest final yields. Researchers then examined ways of improving the productivity of the process. The substrate, benzaldehyde, and the product, L-PAC, as well as the by-products, were found to be toxic to the biomass. Methods examined to reduce toxicity include modification of benzaldehyde dosing regimes, immobilization of biomass or purified enzymes, modification of benzaldehyde solubility and the use of two-phase reaction systems. Various means of modifying metabolism to enhance enzyme activity, relevant metabolic pathways and yield have been examined. Methods investigated include the use of respiratory quotient to influence pyruvate production and induce fermentative activity, reduced aeration to increase PDC activity, and carbohydrate feeding to modify glycolytic enzyme activity. The effect of temperature on L-PAC yield has been examined to identify conditions which provide the optimal balance between L-PAC and benzyl alcohol production, and L-PAC inactivation. However, relatively little work has been undertaken on the effect of medium composition on L-PAC yield.
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PMID:Factors affecting the production of L-phenylacetylcarbinol by yeast: a case study. 1050 Aug 43

Relative peak-height ratios of products to substrates determined by MALDI-TOFMS allow the quantitative analysis of enzyme catalyzed reactions for screening purposes. Two examples were investigated: the first one was a lipase catalyzed reaction which produces 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (MET) using rac-alpha-phenylethylamine (PEA) as substrate. The second one was the pyruvate decarboxylase catalyzed formation of (1R)-1-hydroxy-1-phenyl-2-propanone (PAC) with benzaldehyde (BzA) as substrate. Here the corresponding oximes were analyzed after derivatization using hydroxylamine. The standard curves (r2 = 0.985 for MET, r2 = 0.991 for PAC) were linear over two orders of magnitude for MET and PAC concentrations. After optimization of the sample preparation an average relative standard deviation of 12.5% was obtained in both cases.
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PMID:Quantitation of low molecular mass substrates and products of enzyme catalyzed reactions using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 1108 6

(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.
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PMID:Enzymatic (R)-phenylacetylcarbinol production in benzaldehyde emulsions. 1238 47

A novel assay has been developed for the detection of ( R)-phenylacetylcarbinol, ( R)-PAC, a chiral intermediate in the industrial synthesis of ephedrine. It is the product of a biotransformation of benzaldehyde catalysed by the enzyme pyruvate decarboxylase. The assay, using 2,3,5-triphenyltetrazolium chloride, enables high-throughput photometric analysis of the activity of the enzyme thus avoiding time-consuming chromatographic procedures.
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PMID:High-throughput assay of ( R)-phenylacetylcarbinol synthesized by pyruvate decarboxylase. 1245 21

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.
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PMID:Acetohydroxyacid synthase: a new enzyme for chiral synthesis of R-phenylacetylcarbinol. 1288 23

A pyruvate decarboxylase (PDC) gene from bacterial Zymobacter palmae (Zymopdc) was cloned, characterized, and introduced into Lactococcus lactis via a shuttle vector pAK80 as part of a research strategy to develop an efficient ethanol-producing lactic acid bacteria (LAB). The expression levels of Zymopdc gene in the host, as measured by a colorimetric assay based on PDC catalyzed formation of (R)-phenylacetylcarbinol ((R)-PAC), appeared to be dependent on the strength of corresponding Gram-positive promoters. A constitutive, highly expressed promoter conferred the greatest PDC activity, and an acid-inducible promoter demonstrated acid-inducible expression. The metabolic production of ethanol and other products was examined in flask fermentations. More than eightfold increases in acetaldehyde concentrations were detected in two recombinant strains. However, no detectable differences for ethanol fermentation in these engineered strains were observed compared with that of the strain carrying lacZ reporter.
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PMID:Functional expression of bacterial Zymobacter palmae pyruvate decarboxylase gene in Lactococcus lactis. 1596 4


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