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⁺).     

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.     

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.     

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”.     

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.     

Screening of yeasts for the production of the aroma compound 2-phenylethanol in a molasses-based medium

Fourteen yeast strains were screened for production of 2-phenylethanol from l-phenylalanine with molasses as carbon source. Up to 1 g 2-phenylethanol l^–1 was obtained. Using oleyl alcohol as a second phase for in situ product removal to enhance the production of 2-phenylethanol increased the yield to about 3 g 2-phenylethanol l^–1 at 35 °C. The most productive strains were Kluyveromyces marxianus CBS 600 and CBS 397.     

Enantioselective hydrolysis of insoluble (R,S)-ketoprofen ethyl ester in dispersed aqueous reaction system induced by chiral cyclodextrin

The enantioselective hydrolysis of insoluble (R,S)-ketoprofen ethyl ester to the optically active (S)-ketoprofen was carried out in a dispersed aqueous lipase reaction system induced by the inclusion of chiral cyclodextrins for complexation of the substrate. Hydroxypropyl-beta-cyclodextrin was the most effective chiral selector and disperser giving an enantiomeric excess and conversion yield of 0.99 and 0.49, respectively.     

Identification and Characterization of a Mycobacterial (2R,3R)-2,3-Butanediol Dehydrogenase

Bacterial strain B-009, capable of using racemic 1,2-propanediol (PD), was identified as a rapid-growing member of the genus Mycobacterium. The strain is phylogenetically related to M. gilvum, but has slightly different physiological characteristics. An NAD⁺-dependent enantioselective alcohol dehydrogenase, which acts on R-PD, was purified from the strain. The enzyme was a homodimer of a peptide coded by a 1047-bp gene (mbd1). A highly conserved sequence for medium-chain dehydrogenase/reductases with a preference for secondary alcohols was found in the gene. Hydroxyacetone was produced from R-PD by an enzymatic reaction, indicating that position 2 of the substrate was oxidized. The enzyme activity was highest for (2R,3R)-2,3-butanediol (R,R-BD), enabling the enzyme to be identified as (2R,3R)-2,3-butanediol dehydrogenase (R,R-BD-DH). A homology search revealed M. gilvum, M. vanbaalenii, and M. semegmatis to have ORFs similar to mbd1, suggesting the widespread distribution of genes encoding R,R-BD-DH among mycobacterial strains.     

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.     

New sources of (R)-oxynitrilase: capulin (Prunnus capuli) and mamey (Mammea americana)

(R)-Cyanohydrins were prepared from aldehydes and HCN in diisopropyl ether with high enantiomeric purity (89–98 %ee) by using the defatted meal from capulin (Prunnus capuli) or mamey (Mammea americana) as sources of (R)-oxynitrilase. © Rapid Science Ltd. 1998     

Screening and characterization of yeasts for xylitol production

AIMS: To discover novel naturally occurring xylitol producing yeast species with potential for industrial applications. METHODS AND RESULTS: Exactly 274 strains were cultivated on both solid and liquid screening medium with xylose as the sole carbon resource. Five strains were selected on the basis of significant growth and high degree of xylose assimilation. Their phylogenetic position was confirmed by the PCR-RFLP and sequence analysis of the D1/D2 domain of the 5' end of the large subunit rDNA gene (5'-LSU rDNA). Enzymatic analysis was conducted to compare xylose metabolism in each strain. Candida guilliermondii Xu280 and Candida maltosa Xu316 were found to have high xylose consumption rates and xylitol yields in the batch fermentation under micro-aerobic condition. The effect of the different media with high initial xylose concentration on biosynthesis of xylitol by both strains was investigated. CONCLUSIONS: We have identified Candida spp. strains, which exhibit high levels of xylitol production from xylose suggesting that these may have potential for industrial applications. SIGNIFICANCE AND IMPACTS OF THE STUDY: Microbial species are of importance for xylitol production. Xylitol production involves complicated metabolic regulation including xylose transport, production of key enzymes and cofactor regeneration. Thus, screening of naturally occurring xylose-utilizing micro-organisms is a viable and effective mean to obtain xylitol producing organisms with industrial application. Moreover, the research on selected strains will contribute to a better understanding of regulatory properties of xylose metabolism in different yeasts.     

Screening ofPenicillium species for the production of extracellular glucose oxidase

During screening ofPenicillium species for extracellular glucose oxidase production, a strain ofPenicillium variabile (P16) was selected which released high activities of enzyme into the culture medium. Maximum activity (5.49 U/ml) was after 96 h cultivation in shake-flask culture.M. Petruccioli is with the Dipartimento di Biologia, Difesa e Biotecnologie Agro-Alimentari, University of Basilicata, Via N. Sauro 85, 1-85100 Potenza, Italy. M. Ceccarelli and F. Federici are with the Dipartimento Agrobiologia e Agrochimica, University of Tuscia, Via S.C. de Lellis, 1-01100 Viterbo, Italy.     

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     

(R)-oxynitrilase-catalysed synthesis of chiral silicon-containing aliphatic (R)-ketone-cyanohydrins

Optically active 2-trimethylsilyl-2-hydroxyl-ethylcyanide was prepared by enzymatic enantioselective transcyanation of acetyltrimethylsilane with acetone cyanohydrin in a biphasic system at 35°C and pH 5. (R)-Oxynitrilase from apple seed meal was the best among all the enzymes explored and diisopropyl ether was the most suitable organic phase. Acetyltrimethylsilane was a better substrate of the enzyme than its carbon analogue. The substrate conversion and product enantiomeric excess of 2-trimethylsilyl-2-hydroxyl-ethylcyanide were >99% and >99%, respectively.     

Screening and identification of epiphytic yeasts with potential for biological control of green mold of citrus fruits

Epiphytic yeasts isolated from the surface of citrus fruits, harvested in several orchards in the Souss-Massa-Draa Valley, Agadir, Morocco, were in vivo screened for antagonistic activity against Penicillium digitatum, the causal agent of green mold of citrus. From a total of 245 yeast strains assessed for their biocontrol activity against P. digitatum, fifteen reduced the incidence of disease to less than 50%. The effectiveness of the best selected yeast strains showed that Pichia anomala (YT73), Debaryomyces hansenii (YT22) and Hanseniaspora guilliermondii (YT13) were the most effective, with a reduction of green mold incidence from 65 to ~80%, compared to the control. The identification of the fifteen selected yeast strains was carried out through an integrated approach including phenotypic and genotypic (sequencing of D1/D2 domain of 26S rDNA encoding gene) methods. These 15 selected were identified as: H. guilliermondii, D. hansenii, H. uvarum and P. anomala. The study of the dynamics of two of the best strains, H. guilliermondii and D. hansenii, showed that these strains can grow rapidly, by approximately 2 log units, in citrus fruit wounds. Such rapid growth in wounds indicates that these antagonist yeasts are excellent colonizers of citrus wounds and can thrive on citrus fruits as a substrate.     

A novel enantioselective epoxide hydrolase for (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol

Bacillus sp. Z018, a novel strain producing epoxide hydrolase, was isolated from soil. The epoxide hydrolase catalyzed the stereospecific hydrolysis of (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol. Epoxide hydrolase from Bacillus sp. Z018 was inducible, and (R)-phenyl glycidyl ether was able to act as an inducer. The fermentation conditions for epoxide hydrolase were 35°C, pH 7.5 with glucose and NH₄Cl as the best carbon and nitrogen source, respectively. Under optimized conditions, the biotransformation yield of 45.8% and the enantiomeric excess of 96.3% were obtained for the product (R)-3-phenoxy-1,2-propanediol.     

Purification and properties of polyphenoloxidase of mango peel (Mangifera indica)

Polyphenoloxidase from mango(Mangifera indica) peel was purified to homogeneity by ammonium sulphate fractionation, chromatography on DEAE-Sephadex and gel filtration of Sephadex G-200. The enzyme had an apparent molecular weight of 136,000. Its pH and temperature optimum were 5.4 and 50‡C, respectively. The enzyme possessed catecholase activity and was specific too-dihydroxy phenols. The enzyme also exhibited peroxidase activity. Some non-oxidizable phenolic compounds inhibited the enzyme competitively. High inhibitory effects were also shown by some metal chelators and reducing agents, Mango peel polyphenol oxidase when immobilized onto DEAE Sephadex showed slightly higher Km for catechol and lower pH and temperature optima.     

Screening of plant extracts for antimicrobial activity against bacteria and yeasts with dermatological relevance

There is cumulative resistance against antibiotics of many bacteria. Therefore, the development of new antiseptics and antimicrobial agents for the treatment of skin infections is of increasing interest. We have screened six plant extracts and isolated compounds for antimicrobial effects on bacteria and yeasts with dermatological relevance. The following plant extracts have been tested: Gentiana lutea, Harpagophytum procumbens, Boswellia serrata (dry extracts), Usnea barbata, Rosmarinus officinalis and Salvia officinalis (supercritical carbon dioxide [CO2] extracts). Additionally, the following characteristic plant substances were tested: usnic acid, carnosol, carnosic acid, ursolic acid, oleanolic acid, harpagoside, boswellic acid and gentiopicroside. The extracts and compounds were tested against 29 aerobic and anaerobic bacteria and yeasts in the agar dilution test. U. barbata-extract and usnic acid were the most active compounds, especially in anaerobic bacteria. Usnea CO2-extract effectively inhibited the growth of several Gram-positive bacteria like Staphylococcus aureus (including methicillin-resistant strains - MRSA), Propionibacterium acnes and Corynebacterium species. Growth of the dimorphic yeast Malassezia furfur was also inhibited by Usnea-extract. Besides the Usnea-extract, Rosmarinus-, Salvia-, Boswellia- and Harpagophytum-extracts proved to be effective against a panel of bacteria. It is concluded that due to their antimicrobial effects some of the plant extracts may be used for the topical treatment of skin disorders like acne vulgaris and seborrhoic eczema.     

The effect of growth conditions on production and excretion of extracellular antigens by three ascomycetous yeasts

Ascomycetous yeasts produce extracellular antigens that are almost specific for the species. The antigen production by Hansenula wickerhamii and Stephanoascus ciferrii was independent of the carbon source and was proportional to the final cell density of the cultures. The same was true of chemostat cultures of Stephanoascus ciferrii, irrespective of the dilution rate and whether glucose or ammonia was the limiting nutrient. In cultures of Saccharomyces cerevisiae, however, antigen excretion mainly took place in the late exponential growth phase. Large amounts of antigen were extracted from the cell wall of Saccharomyces cerevisiae. A small amount was detected in the cytoplasm.