Chiral alcohol production by NADH-dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH.

Phenylacetaldehyde reductase (PAR) produced by styrene-assimilating Corynebacterium strain ST-10 was used to synthesize chiral alcohols. This enzyme with a broad substrate range reduced various prochiral aromatic ketones and beta-ketoesters to yield optically active secondary alcohols with an enantiomeric purity of more than 98% enantiomeric excess (e.e.). The Escherichia coli recombinant cells which expressed the par gene could efficiently produce important pharmaceutical intermediates; (R)-2-chloro-1-(3-chlorophenyl)ethanol (28 mg.mL-1) from m-chlorophenacyl chloride, ethyl (R)-4-chloro-3-hydroxy butanoate) (28 mg.mL-1) from ethyl 4-chloro-3-oxobutanoate and (S)-N-tert-butoxycarbonyl(Boc)-3-pyrrolidinol from N-Boc-3-pyrrolidinone (51 mg.mL-1), with more than 86% yields. The high yields were due to the fact that PAR could concomitantly reproduce NADH in the presence of 3-7% (v/v) 2-propanol in the reaction mixture. This biocatalytic process provided one of the best asymmetric reductions ever reported.     

The synthesis and X-ray structure of a chiral rhodium-NHC complex: Implications for the use of NHCs in asymmetric hydroformylation catalysis

An optically active N-heterocyclic carbene (NHC) has been metallated with [RhCl(COD)]₂ to give a rhodium(I) monocarbene substituted complex. This complex has been fully characterized by ¹H and ¹³C NMR spectroscopy and by single-crystal X-ray analysis. Preliminary tests of hydroformylation of styrene catalyzed with this compound have shown that branched to linear aldehyde ratio was rather low 0.67 (40%) and that the enantiomeric excess measured on the corresponding alcohols was only 10%. In the presence of an excess of triphenylphosphine, the ratio of branched to linear aldehyde increased markedly (91%) whereas the enantioselectivity of the reaction remained almost unchanged (12.5%).     

Simple and efficient synthesis of chiral amino alcohols with an amino acid-based skeleton

Sodium bis(2-methoxyethoxy)aluminum hydride, NaAlH₂(OCH₂CH₂OCH₃)₂, commercially known as Vitride® or Red-Al®, enables rapid synthesis of pure optically active N-protected amino alcohols and peptide alcohols in very high yields. The method is very simple and attractive, as it does not require an additional step of N-protected amino acid derivatization and proceeds without the loss of enantiomeric homogeneity.     

Enantioselective epoxidation by an optically active hydroperoxide

Enantioselective epoxidation of prochiral allylic alcohols with optically active 4,6-di-O-acetyl-2,3-dideoxy-α-D -threo-hex-2-enopyranosyl hydroperoxide in the presence of Ti(O-i-Pr)₄ gives chiral epoxy alcohols with moderate enantiomeric excess. © 1993 Wiley-Liss, Inc.     

The application of safe for humans and the environment Polyversum antifungal agent containing living cells of Pythium oligandrum for biotransformation of prochiral ketones

This report presents the whole-cell biotransformation of benzofuranyl-methyl ketone derivatives with the application of Polyversum antifungal agent containing Pythium oligandrum microorganism. Stereochemistry of the reduction of prochiral substrates was modified by the bioconversion conditions (concentration of reagents, a source of the carbon atom, biotransformation medium). In optimized conditions enantioselective process was noted. Secondary alcohols with excellent enantiomeric purity and high yields were obtained. The enantiomeric excess and conversion degree of 1-(benzofuran-2-yl)ethanol, 1-(7-ethylbenzofuran-2-yl)ethanol and 1-(3,7-dimethylbenzofuran-2-yl)ethanol were 99%/98.1%, 94%/94.4% and 99%/72.6%, respectively. In the presence of P. oligandrum, one of the enantiotopic hydrides of the dihydropyridine ring coenzyme is selectively transferred to a re side of the prochiral carbonyl group to give products with S configuration. This study demonstrates an inexpensive, eco-friendly approach in synthesis of optically pure benzofuran derivatives and can be an interesting alternative to organocatalysis. Furthermore, this method can be used in biotechnology processes due to its good chemical performance and a high degree of product isolation.     

On the kinetics of the enzyme-catalyzed hydrolysis of axial chiral alkyl allenecarboxylates: preparation of optically active R-(−)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid and its optically pure S-(+)-methylester

Pig liver esterase (PLE) was used for the preparation of optically active alkyl allenecarboxylates with axial chirality. Free and immobilized enzymes were used as biocatalysts for the kinetic resolution of racemic ester substrates. Whereas the biotransformations using the free biocatalyst resulted in moderately to high enantiomeric ratios, the immobilization significantly decreased the E-value. The reaction conditions were optimized with respect to the enantiomeric ratio and scaled up. The enantiomeric ratio (E-value) was thereby enhanced by a factor of four to E=60. Under optimized conditions (free enzyme, addition of acetone as a cosolvent and Triton X-100 as an emulgator) in a preparative scale biotransformation, 282 mg of optically pure S-(+)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid methylester (96% ee, 82% yield) and 257 mg of R-(−)-2-ethyl-4-phenyl-2,3-hexadiene-carboxylic acid (83% ee, 80% yield) could be synthesized from the racemic substrate.     

Lipases and whole cell biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and its ester

A wide spectrum of commercially available lipases and microbial whole cells catalysts were tested for biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 and its butyryl ester. The best results were achieved for biocatalytic hydrolysis of ester: 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 performed by lipase from Candida cylindracea, what gave optically active products with 85% enantiomeric excess, 50% conversion degree and enantioselectivity 32.9 for one pair of enantiomers. Also enzymatic systems of Penicillium minioluteum and Fusarium oxysporum were able to hydrolyze tested compound with high enantiomeric excess (68–93% ee), enantioselectivity (44 for one pair of enantiomers) and conversion degree about 50–55%. Enzymatic acylation of hydroxyphosphinate was successful in case when porcine pancreas lipase was used. After 4 days of biotransformation the conversion reaches 45% but the enantiomeric enrichment of the isomers mixture do not exceed 43%. Obtained chiral compounds are valuable derivatizing agents for spectroscopic (NMR) evaluation of enantiomeric excess for particular compounds (e.g. amino acids).     

Efficient Production of (R)-2-Hydroxy-4-Phenylbutyric Acid by Using a Coupled Reconstructed d-Lactate Dehydrogenase and Formate Dehydrogenase System

Background

(R)-2-Hydroxy-4-phenylbutyric acid [(R)-HPBA] is a key precursor for the production of angiotensin-converting enzyme inhibitors. However, the product yield and concentration of reported (R)-HPBA synthetic processes remain unsatisfactory.

Methodology/Principal Findings

The Y52L/F299Y mutant of NAD-dependent d-lactate dehydrogenase (d-nLDH) in Lactobacillus bulgaricus ATCC 11842 was found to have high bio-reduction activity toward 2-oxo-4-phenylbutyric acid (OPBA). The mutant d-nLDH^Y52L/F299Y was then coexpressed with formate dehydrogenase in Escherichia coli BL21 (DE3) to construct a novel biocatalyst E. coli DF. Thus, a novel bio-reduction process utilizing whole cells of E. coli DF as the biocatalyst and formate as the co-substrate for cofactor regeneration was developed for the production of (R)-HPBA from OPBA. The biocatalysis conditions were then optimized.

Conclusions/Significance

Under the optimum conditions, 73.4 mM OPBA was reduced to 71.8 mM (R)-HPBA in 90 min. Given its high product enantiomeric excess (>99%) and productivity (47.9 mM h⁻¹), the constructed coupling biocatalysis system is a promising alternative for (R)-HPBA production.     

Resolution of biotin intermediate lactone by enzyme-catalyzed stereoselective lactonization in organic solvent

A strain of Aspergillus oryzae WZ007 producing intracellular lipase with preference for the formation of the d-biotin intermediate lactone ((3aS,6aR)-tetrahydro-1,3-dibenzylhexahydro-1H-Furo[3,4-d]imidazole-2,4-dione) 1 was screened from soil samples. The microbial cells fermented by the strain were used as biocatalysts after lyophilization, to resolve the racemic biotin intermediate lactone 1 by enantioselective lactonization in organic solvents. The enantioselective lactonization afforded optically active lactone 1 with high enantiomeric excess (e.e. ≥ 98%) and conversion ratio (c ≥ 40%) when the biotransformation was carried out in dichloromethane. The e.e. of product was up to 99% after purification by inducing crystallization in acetone and basically satisfied the industrial application. The biosynthesis has been employed to produce kilograms supplies of optically active lactone 1 with high optical purity.     

Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor: promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction

An asymmetric hydrogen-transfer biocatalyst consisting of mutated Rhodococcus phenylacetaldehyde reductase (PAR) or Leifsonia alcohol dehydrogenase (LSADH) was applied for some water-soluble ketone substrates. Among them, 4-hydroxy-2-butanone was reduced to (S)/(R)-1,3-butanediol, a useful intermediate for pharmaceuticals, with a high yield and stereoselectivity. Intact Escherichia coli cells overexpressing mutated PAR (Sar268) or LSADH were directly immobilized with polyethyleneimine or 1,6-diaminehexane and glutaraldehyde and evaluated in a batch reaction. This system produced (S)-1,3-butanediol [87% enantiomeric excess (e.e.)] with a space time yield (STY) of 12.5 mg h⁻¹ ml⁻¹ catalyst or (R)-1,3-butanediol (99% e.e.) with an STY of 60.3 mg h⁻¹ ml⁻¹ catalyst, respectively. The immobilized cells in a packed bed reactor continuously produced (R)-1,3-butanediol with a yield of 99% (about 49.5 g/l) from 5% (w/v) 4-hydroxy-2-butanoate over 500 h.     

Debaryomyces hansenii as a new biocatalyst in the asymmetric reduction of substituted acetophenones

Chiral secondary alcohols are convenient mediator for the synthesis of biologically active compounds and natural products. In this study fifteen yeast strains belonging to three food originated yeast species Debaryomyces hansenii, Saccharomyces cerevisiae and Hanseniaspora guilliermondii were tested for their capability for the asymmetric reduction of acetophenone to 1-phenylethanol as biocatalyst microorganisms. Of these strains, Debaryomyces hansenii P1 strain showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to the corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high conversion rates. This is the first report on the enantioselective reduction of acetophenone by D. hansenii P1 from past?rma, a fermented Turkish meat product. The preparative scale asymmetric bio reduction of 3-methoxy acetophenone 1g by D. hansenii P1 gave (R)-1-(3-methoxyphenyl) ethanol 2g 82% yield, and >99% enantiomeric excess. Compound 2g can be used for the synthesis of (+)-NPS-R-568 [3-(2-chlorophenyl)-N-[(1R)-1-(3-methoxyphenly) ethyl] propan-1-amine] which have a great potential for the treatment of primary and secondary hyper-parathyroidism. In addition, D. hansenii P1 successfully reduced acetophenone derivatives. This study showed that this yeast can be used industrially to produce enantiomerically pure chiral secondary alcohols, which can be easily converted to different functional groups.     

Microbial oxidation of methanol: Properties of crystallized alcohol oxidase from a yeast, Pichia sp

Alcohol oxidase (alcohol:oxygen oxidoreductase) was crystallized from a methanolgrown yeast, Pichia sp. The crystalline enzyme is homogenous as judged from polyacrylamide gel electrophoresis. Alcohol oxidase catalyzed the oxidation of short-chain primary alcohols (C₁ to C₆), substituted primary alcohols (2-chloroethanol, 3-chloro-1-propanol, 4-chlorobutanol, isobutanol), and formaldehyde. The general reaction with an oxidizable substrate is as follows: Primary alcohol + O₂ → aldehyde + H₂O₂ Formaldehyde + O₂ → formate + H₂O₂. Secondary alcohols, tertiary alcohols, cyclic alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized. The K_m values for methanol and formaldehyde are 0.5 and 3.5 mm, respectively. The stoichiometry of substrate oxidized (alcohol or formaldehyde), oxygen consumed, and product formed (aldehyde or formate) is 1:1:1. The purified enzyme has a molecular weight of 300,000 as determined by gel filtration and a subunit size of 76,000 as determined by sodium dodecyl sulfate-gel electrophoresis, indicating that alcohol oxidase consists of four identical subunits. The purified alcohol oxidase has absorption maxima at 460 and 380 nm which were bleached by the addition of methanol. The prosthetic group of the enzyme was identified as a flavin adenine dinucleotide. Alcohol oxidase activity was inhibited by sulfhydryl reagents (p-chloromercuribenzoate, mercuric chloride, 5,5′-dithiobis-2-nitrobenzoate, iodoacetate) indicating the involvement of sulfhydryl groups(s) in the oxidation of alcohols by alcohol oxidase. Hydrogen peroxide (product of the reaction), 2-aminoethanol (substrate analogue), and cupric sulfate also inhibited alcohol oxidase activity.     

Simple and efficient synthesis of chiral amino alcohols with an amino acid-based skeleton

Sodium bis(2-methoxyethoxy)aluminum hydride, NaAlH₂(OCH₂CH₂OCH₃)₂, commercially known as Vitride® or Red-Al®, enables rapid synthesis of pure optically active N-protected amino alcohols and peptide alcohols in very high yields. The method is very simple and attractive, as it does not require an additional step of N-protected amino acid derivatization and proceeds without the loss of enantiomeric homogeneity.     

Stereospecific oxidation of aliphatic alcohols catalyzed by galactose oxidase

The stereospecificity of galactose oxidase (EC 1.1.3.9) from Dactylium dendroides in the oxidation of simple three-carbon alcohols has been examined. The enzyme oxidizes glycerol to optically pure S(?)glyceraldehyde. In addition to this prochiral stereospecificity, galactose oxidase also exhibits enantiomeric stereospecificity in the oxidation of 3-halo-1,2-propanediols: the R isomer appears to be a better substrate than its S counterpart. The above stereochemistry of galactose oxidase-catalyzed oxidation of “unnatural” substrates, non-sugar alcohols, can be predicted on the basis of the conformation of the natural substrate of the enzyme, D-galactose.     

Effects of Light on the Microcystin Content of Microcystis Strain PCC 7806

Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 μmol of photons m⁻² s⁻¹ on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell⁻¹ and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 μmol of photons m⁻² s⁻¹, the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 μmol of photons m⁻² s⁻¹ than at 10 μmol of photons m⁻² s⁻¹ and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.     

Development of an improved phenylacetaldehyde reductase mutant by an efficient selection procedure

Chiral alcohols are valuable as diverse chemicals and synthetic intermediate materials. Phenylacetaldehyde reductase (PAR) is an enzyme that converts a wide variety of ketones into chiral alcohols with high optical purity. When an alcohol such as 2-propanol is used as a hydrogen donor, PAR itself will also mediate the regeneration of the coenzyme NADH in situ. Perceiving a capacity for improvement, we sought to develop a PAR that is able to convert higher concentrations of substrates in the presence of high concentrations of 2-propanol. The selection procedure for mutants was re-examined and a procedure able to select an effective amino acid substitution was established. Two advantageous amino acid substitutions were successfully selected using the procedure. When high-concentration substrate conversion reaction was subjected with a mutant that integrated both the two amino acid substitutions, near-complete conversions of m-chlorophenacyl chloride (m-CPC) (2.1 mmol/ml) and ethyl 4-chloro-3-oxobutanoate (ECOB) (1.9 mmol/ml) were achieved.     

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost‐effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co‐immobilized them on modified poly‐p‐hydroxybutyrate synthase (PhaC)‐inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled‐coil interaction. Enzyme‐loaded particles could be easily purified by centrifugation. Total conversion of 4'‐chloroacetophenone to (S)‐4‐chloro‐α‐methylbenzyl alcohol could be accomplished using enzyme‐loaded particles, catalytic amounts of NAD⁺ and formate as substrates for FDH. Chiral GC‐MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost‐effective alternative to coupled reactions using purified enzymes.     

河南省烤烟大田生长期光合有效辐射空间变异性

地统计学是研究空间变异性的一种统计方法。结合河南省1950—2000年整编气象数据,计算出烟草各生长阶段的光合有效辐射量,并采用地统计学与GIS相结合的方法,研究了大尺度下河南省烤烟种植区烟草各生长阶段的光合有效辐射的空间变异性及分布特征。经典统计学分析表明,光合有效辐射在河南省境内可以较好地拟合成正态分布,满足地统计学所要求的平稳假设条件。其境内光合有效辐射中变异系数最大的是5月份,为9.61%,最小的是7月份,为3.92%。地统计学表明,河南省烟草大田生长期光合有效辐射可以拟合成为高斯模型,自相关距离为1719300.00 m,块金效应C0/sill为21.36%,具有强空间相关性。河南省烟草大田生长期光合有效辐射量987—1024 J/m2的低值区占研究区总面积的19.37%,主要分布在南阳、信阳、洛阳、三门峡、平顶山;光合有效辐射量1024—1060 MJ/m2的区域分布在三门峡、洛阳、平顶山、南阳,驻马店、信阳、洛河,占研究区总面积的34.50%;光合有效辐射量1060—1097 MJ/m2分布在济源、洛阳、郑州、平顶山、许昌、漯河、驻马店、周口、商丘、三门峡、新乡、安阳,占研究区总面积的19.98%;光合有效辐射量1097—1133 MJ/m2的次高值区占研究区总面积的26.15%,主要分布在开封、鹤壁、焦作、新乡、安阳、濮阳、郑州、济源和商丘;光合有效辐射量1133—1170 MJ/m2的高值区分布在濮阳、安阳,占研究区总面积的3.02%。研究表明,采用地统计学分析结合GIS技术,可以较方便地分析大尺度的光合有效辐射量的空间变异规律,从而为提高烤烟等大田作物的田间管理水平与品质产量提供精准的数据。     

Growth and metabolism of the obligate photolithotroph Chlorobium thiosulfatophilum in the presence of added organic nutrients

Growth of Chlorobium vibrioforme f. thiosulfatophilum NCIB 8327 could be monitored by measurement of turbidity (E₆₀₀); absorbance at 745 and 665 nm; increase in methanol-extractable pigment (E₆₆₀); fixation of ¹⁴CO₂; and titration of thiosulphate and sulphide in the medium. Growth could be inhibited by formate, methionine, tryptophan, tyrosine, threonine, serine and glycine, but not by 14 other amino acids, shikimic acid, some alcohols, sugars or acetate. Inhibition could some-times be relieved by the presence of other amino acids. This was probably partly due to restoration of normal internal amino acid requirements by “feeding”, and partly because uptake of amino acids appeared to show some competition for two or more low specificity uptake systems. Numerous ¹⁴C-labelled amino acids, formate and glucose were shown to be photoassimilated by Chlorobium, and the labelling patterns obtained provided information on its pathways of intermediary biosynthesis. Growth inhibition by threonine could be related to the probable presence of a normal branched pathway for the synthesis of the aspartate family of amino acids, with an aspartokinase enzyme subject to strong inhibition by threonine and lysine, separately and in combination.     

Competing formate- and carbon dioxide-utilizing prokaryotes in an anoxic methane-emitting fen soil

Methanogenesis in wetlands is dependent on intermediary substrates derived from the degradation of biopolymers. Formate is one such substrate and is stimulatory to methanogenesis and acetogenesis in anoxic microcosms of soil from the fen Schlöppnerbrunnen. Formate dissimilation also yields CO₂ as a potential secondary substrate. The objective of this study was to resolve potential differences between anaerobic formate- and CO₂-utilizing prokaryotes of this fen by stable isotope probing. Anoxic soil microcosms were pulsed daily with low concentrations of [¹³C]formate or ¹³CO₂ (i.e., [¹³C]bicarbonate). Taxa were evaluated by assessment of 16S rRNA genes, mcrA (encoding the alpha-subunit of methyl-coenzyme M reductase), and fhs (encoding formyltetrahydrofolate synthetase). Methanogens, acetogens, and formate-hydrogen lyase-containing taxa appeared to compete for formate. Genes affiliated with Methanocellaceae, Methanobacteriaceae, Acetobacteraceae, and Rhodospirillaceae were ¹³C enriched (i.e., labeled) in [¹³C]formate treatments, whereas genes affiliated with Methanosarcinaceae, Conexibacteraceae, and Solirubrobacteraceae were labeled in ¹³CO₂ treatments. [¹³C]acetate was enriched in [¹³C]formate treatments, but labeling of known acetogenic taxa was not detected. However, several phylotypes were affiliated with acetogen-containing taxa (e.g., Sporomusa). Methanosaetaceae-affiliated methanogens appeared to participate in the consumption of acetate. Twelve and 58 family-level archaeal and bacterial 16S rRNA phylotypes, respectively, were detected, approximately half of which had no isolated representatives. Crenarchaeota constituted half of the detected archaeal 16S rRNA phylotypes. The results highlight the unresolved microbial diversity of the fen Schlöppnerbrunnen, suggest that differing taxa competed for the same substrate, and indicate that Methanocellaceae, Methanobacteriaceae, Methanosarcinaceae, and Methanosaetaceae were linked to the production of methane, but they do not clearly resolve the taxa responsible for the apparent conversion of formate to acetate.