Site-specific and asymmetric hydrolysis of prochiral 2-phenyl-1,3-propanediol diacetate by a bacterial esterase from an isolated strain

Bacillus cereus 809A and Burkholderia sp. 711C were isolated from soil. These strains demonstrate hydrolysis activity towards prochiral 2-phenyl-1,3-propanediol diacetate and accumulated the corresponding chiral monoacetates into the reaction mixture. When 2-phenyl 1,3-propanediol diacetate was used as a substrate, the produced monoacetates with Burkholderia sp. 711C were obtained in a racemic form but that produced by Bacillus cereus 809A showed an excess of the (S)-form. The resting cell reaction revealed that for Bacillus cereus 809A, there was an enrichment of one of the enantiomers of the monoacetate such that the enantiomeric excess (e.e.) of the (S)-form was over 95%. The purified enzyme from Bacillus cereus 809A hydrolyzed diacetate to monoacetate, and the e.e. value of the (S)-form increased by prolonged reaction in a way similar to the resting cell reaction. From N-terminal amino acids, this esterase is conserved in some strains of Bacillus for which the genomic sequences have been reported.     

Stereoselective enzymatic hydrolysis of 2-cyclohexyl-and 2-phenyl-1,3-propanediol diacetate in biphasic systems

Summary A key intermediate (S(–) 2-cyclohexyl-1,3-propanediol monoacetate) was made with high optical purity for the total synthesis of a new angiotensin converting enzyme inhibitor, Fosinopril. The stereoselective hydrolysis of 2-cyclohexyl-1,3-propanediol diacetate (I) and 2-phenyl-1,3-propanediol diacetate (II) was carried out with lipases. Among various lipases evaluated, only porcine pancreatic lipase (PPL) and Chromobacterium viscosum lipase demonstrated efficient conversion and gave the desired enantiomer of monoacetate. In aqueous solution, the desired S(–) monoacetate exhibited an optical purity of 65%–80% (30%–60% enantiomeric excess [e.e.]). However, when the same reactions were conducted in a biphasic system, the product S(–) monoacetate exhibited an optical purity of 99%–100% (98%–100% e.e.). The high purity product was achieved with 65 mol% yield at 1% substrate concentration. Among various solvents evaluated in biphasic systems, efficient hydrolysis was achieved in toluene, cyclohexane, and trichloro-trifluoroethane. The crude PPL was partially purified and two lipase fractions (A and B) were identified. Lipases A and B had a molecular mass of 38 000 and 40 000 daltons, respectively, and both were found to catalyze the hydrolysis of I and II to the appropriate monoacetate in a biphasic system. Offprint requests to: R. N. Patel     

Isolation of a bacterium assimilating (R)-3-chloro-1,2-propanediol and production of (S)-3-chloro-1,2-propanediol using microbial resolution

A bacterium capable of assimilating 3-chloro-1,2-propanediol was isolated from soil by enrichment culture. The strain was identified as Alcaligenes sp. by taxonomic studies. The crude extracts of the cells had dehalogenating activities and converted various halohydrins to the corresponding epoxides. 3-Chloro-1,2-propanediol was degraded stereospecifically by the strain, liberating chloride ion. The residual isomer was found to be the (S)-form (99.4% enantiomeric excess). (S)-3-Chloro-1,2-propanediol was obtained from the racemate by use of this strain in 38% yield, and (S)-glycidol (99.4% enantiomeric excess) was subsequently synthesized from the obtained (S)-3-chloro-1,2-propanediol by alkaline treatment.     

Production of highly optically active (R)-3-chloro-1,2-propanediol using a bacterium assimilating the (S)-isomer

A bacterium that assimilates (S)-3-chloro-1,2-propanediol [monochlorohydrin (MCH)] was isolated from soil by enrichment culture. The bacterium was identified as Pseudomonas sp. by taxonomic studies. The strain grew in a medium containing racemic MCH as a source of carbon and degraded (S)-MCH stereoselectively, liberating chloride ions. The residual isomer was the (R)-form [99.5% enantiomeric excess (ee)], which was obtained from the racemate in a final yield of 36% by using this strain. Subsequently, highly optically active (R)-glycidol (GLD) (99.3% ee) was prepared from the (R)-MCH obtained by reaction in alkaline solution. The cell-free extracts of the cells had both dehalogenating and epoxide-opening activities, which converted various halohydrins to the corresponding epoxides and epoxides to the corresponding diols, respectively. Correspondence to: T. Suzuki     

Microbial production of (S)-1-phenyl-1,3-propanediol by stereospecific reduction of 3-hydroxy-1-phenylpropane-1-one

A novel microbial method of synthesizing (S)-1-phenyl-1,3-propanediol [(S)-PPD] was developed in this study. Our laboratory stock cultures were screened for microorganisms that stereospecifically produced (S)-PPD from 3-hydroxy-1-phenylpropane-1-one (HPPO) using an intact cell system. Of the 828 strains examined (321 bacteria, 233 yeasts and 274 molds), certain strains of Williopsis saturnus var. mrakii and Cryptococcus albidus were found to produce (S)-PPD with over 99% enantiomeric excess (e.e.). Screening identified W. saturnus var. mrakii AJ-5620 as the most productive strain, and this strain was used for further experiments. The (S)-PPD-producing reaction using intact W. saturnus var. mrakii AJ-5620 cells was carried out by successive feeding of HPPO. A total (S)-PPD yield of 9.9 g/l was produced in 20 h. The molar yield was 81% and the optical purity of the (S)-PPD produced was over 99% e.e.     

Microbial asymmetric oxidation of 2-butyl-1,3-propanediol

Microbial asymmetric oxidation of 2-butyl-1,3-propanediol was investigated for an efficient synthesis of S- and R-enantiomers of 2-hydroxymethylhexanoic acid (2-HMHA). From an intensive survey of the stocked bacterial strains, Acetobacter pasteurianus IAM 12073 and Pseudomonas putida IFO 3738 were found to show the highest S- and R-2-HMHA-producing activity, respectively. Under optimized conditions, A. pasteurianus (351 mg dry cell weight) and P. putida (642 mg dry cell weight) cells produced 12.0 g l⁻¹ S-2-HMHA with 89% enantiomeric excess (e.e.) at 24 h of incubation and 5.1 g l⁻¹ R-2-HMHA with 94% e.e. at 35 h of incubation from 2-butyl-1,3-propanediol.     

Stereoselective enzymatic hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol diacetate ester in a biphasic system

Summary A key chiral intermediate lactol(3)[3aS (3a,4,7,7a)]-hexahydro-4,7-epoxy-isobenzofuran-1 (3H)-one was prepared for the total synthesis of a new thromboxane antagonist. The stereoselective hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol, diacetate ester (1) to the corresponding chiral monoacetate ester (2) was carried out with lipases, among which Amano P-30 lipase from Pseudomonas sp. was most effective since it gave the desired enantiomer of monoacetate ester. A yield of 75 mol% and optical purity of >99% was obtained when the reaction was conducted in a biphasic system with 10% toluene at 5 g/l of the substrate. Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (five cycles) without loss of enzyme activity, productivity or optical purity. The reaction process was scaled-up to 80 1 (400 g substrate) and monoacetate (2) was isolated in 80 mol% yield with 99.3% optical purity as determined by chiral HPLC and nuclear magnetic resonance (NMR) analysis. A gas chromatography of 99.5% and specific rotation, []_D of -7.6° was obtained. The chiral monoacetate ester (2) was oxidized to its corresponding aldehyde and subsequently hydrolyzed to give lactol (3).     

Synthesis of Optically Active Diols by Escherichia coli Transformant Cells that Express the Glycerol Dehydrogenase Gene of Hansenula polymorphaDL‐1

Chiral alcohols are useful as intermediates for the synthesis of drugs. In the production of chiral alcohols, microbial enzymes are promising since high optical purity is required. Under these conditions, the reaction by resting cells is more convenient and inexpensive than by an enzyme reaction. Chiral 1,2‐propanediol and 2,3‐butanediol were obtained using cells expressing the enzyme which demonstrated high stereospecificity. By means of recombinant cells expressing the glycerol dehydrogenase of Hansenula polymorpha DL‐1, the medium was enriched with (S)‐1,2‐propanediol (98 % enantiometric excess, e.e.) during a 24‐h incubation, whereas the (R)‐form was removed from 100 mM of the racemate (R:S = 1:1). For an asymmetric reduction, a recombinant was constructed which also expressed the glucose dehydrogenase gene of Bacillus subtilis origin as an NADH reproducer. In the resting cell reaction, the pH control at 7.5 promoted the conversion from ketone to alcohol. (2R,3R)‐2,3‐butanediol (e.e. > 99.9 %; 308 mM) was produced from 800 mM acetoin (R:S = 3:4); (R)‐1,2‐propanediol (e.e. > 99.9 %; 550 mM) from 800 mM acetol in a 33‐h incubation by the addition of glucose and ketone with pH control. In this reaction, (S)‐forms of both 2,3‐butanediol and 1,2‐propanediol were not produced. The pH control and feeding of the substrates were uncomplicated. The production process of the chiral alcohol by the recombinants which expresses glycerol dehydrogenase proved convenient.     

Production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile by the combination of nitrile hydratase and stereoselective amidase in Rhodococcus equi TG328

A new soil isolate, tentatively identified as Rhodococcus equi TG328, was found to be effective in the production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile. The conversion is catalysed by two enzymes. First, a nitrile hydratase converts the (R,S)-nitrile to (R,S)-2-phenylpropionamide. Second, a stereoselective amidase converts the S-(+)-amide to S-(+)-2-phenylpropionic acid. Conditions for optimal enzyme production and accumulation of S-(+)-2-phenylpropionic acid by resting cells were studied. The reaction of resting cells for 30 h at 10° C with (R,S)-2-phenylpropionitrile resulted in the production of 100 g of S-(+)-2-phenylpropionic acid per litre of reaction mixture. The enantiometric excess of the purified S-(+)-2-phenylpropionic acid was 99.4%. The amount of S-(+)-2-phenylpropionic acid accumulated was enhanced by lower reaction temperatures. In addition, unreacted R-(–)-2-phenylpropionamide with 99.0% enantiometric excess was isolated. Correspondence to: T. Nagasawa     

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.     

Evaluation of Nutritive Value of Glycol Esters as Energy Source for Growing Chicks and Rats

Nutritive value of 4 glycol esters, i.e. ethanediol diacetate, 1,2-propanediol diacetate, 1,3-butanediol diacetate and 1,3-butanediol dioctylate, was estimated biologically by feeding the esters to growing chicks and rats. Energy in the esters taken by both chicks and rats was well utilized, though feed intake of the diets containing the esters at high level tended to decrease. Bitter taste of the esters was suspected to be related to low appetite. The acetates were somewhat volatile and released free acetic acid in the diet during storage. These properties of the acetates makes their use for dietary energy source difficult in practical condition.     

Asymmetric oxidation of 1,3-propanediols to chiral hydroxyalkanoic acids by Rhodococcus sp. 2N

Rhodococcus sp. 2N was found as a 1,3-propanediols-oxidizing strain from soil samples through enrichment culture using 2,2-diethyl-1,3-propanediol (DEPD) as the sole carbon source. The culture condition of the strain 2N was optimized, and the highest activity was observed when 0.3% (w/v) DEPD was added in the culture medium as an inducer. Chiral HPLC analysis of the hydroxyalkanoic acid converted from 2-ethyl-2-methyl-1,3-propanediol (EMPD) revealed that the strain 2N catalyzed the (R)-selective oxidation of EMPD. The reaction products and intermediates from DEPD and EMPD were identified by nuclear magnetic resonance analyses, and the results suggested that only one hydroxymethyl group of the propanediols was converted to carboxy group via two oxidation steps. Under optimized conditions and after a 72-h reaction time, the strain 2N produced 28 mM (4.1 g/L) of 2-(hydroxymethyl)-2-methylbutanoic acid from EMPD with a molar conversion yield of 47% and 65% ee (R).     

Whole‐cell biocatalytic of Bacillus cereus WZZ006 strain to synthesis of indoxacarb intermediate: (S)‐5‐Chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester

In this study, a newly isolated strain screened from the indoxacarb‐rich agricultural soils, Bacillus cereus WZZ006, has a high stereoselectivity to racemic substrate 5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester. (S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester was obtained by bio‐enzymatic resolution. After the 36‐hour hydrolysis in 50‐mM racemic substrate under the optimized reaction conditions, the e.e._s was up to 93.0% and the conversion was nearly 53.0% with the E being 35.0. Therefore, B cereus WZZ006 performed high‐level ability to produce (S)‐5‐chloro‐1‐oxo‐2,3‐dihydro‐2‐hydroxy‐1H‐indene‐2‐carboxylic acid methyl ester. This study demonstrates a new biocatalytic process route for preparing the indoxacarb chiral intermediates and provides a theoretical basis for the application of new insecticides in agricultural production.     

Scale-up of the enantioselective reaction for the enzymatic resolution of (R,S)-ibuprofen

The reaction for the resolution of (R,S)-ibuprofen was scaled-up to yield gram quantities of (S)-ibuprofen. This was accomplished through two enantioselective reactions each catalysed by Novozym 435. In the first reaction, starting from 300 g of racemic ibuprofen, 88.9 g of enantioenriched (S)-ibuprofen with an enantiomeric excess of the order of 85% were produced. In the subsequent reaction, 75 g of the 85 % e.e. material were utilized to produce 38.4 g of (S)-ibuprofen with an enantiomeric excess of 97.5 %.     

L-Cystathionine as a Component of Ezomycins A1 and B1 from a Streptomyces

(1R,2S)-1-(3′-Chloro-4′-methoxyphenyl)-1,2- propanediol (Trametol, 3), a metabolite of the fungus Trametes sp. IVP-F640 and Bjerkandera sp. BOS55, was synthesized by employing Sharpless asymmetric dihydroxylation as the key step. Similarly, the (1R,2S)-isomer of 1-(3′,5′-dichloro-4′-methoxyphenyl)-1,2-propanediol (4), another metabolite of Bjerkandera sp. BOS55, was synthesized by asymmetric dihydroxylation.     

An enantioselective NADP+-dependent alcohol dehydrogenase responsible for cooxidative production of (3S)-5-hydroxy-3-methyl-pentanoic acid

A soil bacterium, Mycobacterium sp. B-009, is able to grow on racemic 1,2-propanediol (PD). The strain was revealed to oxidize 3-methyl-1,5-pentanediol (MPD) to 5-hydroxy-3-methyl-pentanoic acid (HMPA) during growth on PD. MPD was converted into an almost equimolar amount of the S-form of HMPA (S-HMPA) at 72%ee, suggesting the presence of an enantioselective MPD dehydrogenase (MPD-DH). As expected, an NADP⁺-dependent alcohol dehydrogenase, which catalyzes the initial step of MPD oxidation, was detected and purified from the cell-free extract. This enzyme was suggested to be a homodimeric medium-chain alcohol dehydrogenase/reductase (MDR). The catalytic and kinetic parameters indicated that MPD is the most suitable substrate for the enzyme. The enzyme was encoded by a 1047-bp gene (mpd1) and several mycobacterial strains were found to have putative MDR genes similar to mpd1. In a phylogenetic tree, MPD-DH formed an independent clade together with the putative MDR of Mycobacterium neoaurum, which produces opportunistic infections.     

Enzymatic asymmetrization of prochiral 2-benzyl-1,3-propanediol through esterification in solvent media

The enzymatic esterification of the prochiral substrate, 2-benzyl-1,3-propanediol, has been studied in solvent media. Among the five tested lipases, Lipozyme and Novozym 435 led to higher reaction rates. Novozym 435 catalyzed faster reactions at low water activity and in solvents having log P above 2. However, the two positions of the diol, pro-(R) and pro-(S), led to the same reaction rate trends and no prochiral selectivity was obtained. When using Lipozyme in toluene, the reaction rates for the formation of both (R) and (S) products presented an optimum at a water activity of 0.22. In this case, the prochiral selectivity increased with the water activity, from a value of 5 at a _w < 0.01, to a value of 8 at a _w = 0.22, at which point it remained constant.     

Bioproduction of chiral mandelate by enantioselective deacylation of α-acetoxyphenylacetic acid using whole cells of newly isolated Pseudomonas sp. ECU1011

Substrate-directed screening was carried out to find bacteria that could deacylate O-acetylated mandelic acid from environmental samples. From more than 200 soil isolates, we identified for the first time that Pseudomonas sp. ECU1011 biocatalytically deacylated (S)-α-acetoxyphenylacetic acid with high enantioselectivity (E > 200), yielding (S)-mandelic acid with 98.1% enantiomeric excess (ee) at a 45.5% conversion rate. The catalytic deacylation of (S)-α-acetoxyphenylacetic acid by the resting cell was optimized using a single-factor method to yield temperature and pH optima of 30°C and 6.5, respectively. These optima help to reduce the nonselective spontaneous hydrolysis of the racemic substrate. It was found that substrate concentrations up to 60 mM could be used. 2-Propanol was used as a moderate cosolvent to help the substrate disperse in the aqueous phase. Under optimized reaction conditions, the ee of the residual (R)-α-acetoxyphenylacetic acid could be improved further, to greater than 99%, at a 60% conversion rate. Furthermore, using this newly isolated strain of Pseudomonas sp. ECU1011, three kinds of optically pure analogs of (S)-mandelic acid and (R)-α-acetoxyphenylacetic acid were successfully prepared at high enantiomeric purity.     

Asymmetric reduction of aryl ketones with a new isolate Rhodotorula sp. AS2.2241

A yeast strain, Rhodotorula sp. AS2.2241, capable of reducing acetophenone and α-bromoacetophenone with high stereoselectivity, was isolated from soil samples through a novel screening procedure in which acetophenone was supplied in vapor state as the sole carbon and energy source. The biosynthesis of the ketone reductase in the yeast cells reached a maximum of 41.0 U/l at 20 h of cultivation. The reductase isolated from the Rhodotorula sp. cells was partially purified by 52.6-fold through a single column chromatography of DEAE–cellulose. The catalytic performance of the partially purified reductase was examined, and the highest activity was observed at pH 6.5 and 50 °C. The short-chain alkyl aldehydes such as acetaldehyde and those aldehydes or ketones with a benzoyl group were found to be good substrates for the reductase. In the preparative bioreductions of 50 mM acetophenone and 2 mM α-bromoacetophenone using resting cells of Rhodotorula sp. AS2.2241, (S)-(−)-1-phenylethanol (>99.5% enantiomeric excess (e.e.), 34.7% yield) and (R)-(−)-2-bromo-1-phenylethanol (>99.9% e.e., 19.9% yield) were obtained, respectively.     

The headspace-GC/MS: Alternative methodology employed in the bioreduction of (4S)-(+)-carvone mediated by human skin fungus

Abstract

The development of more efficient and environmentally friendly analytical methods represents a current focus for the fine chemical industry. In particular, microscale methodologies that are free of solvents/reagents. The headspace-GC/MS (HS-GC) methodology was employed in this study as a tool for monitoring a biocatalysed reaction of (4S)-(+)-carvone using Phoma sp., a filamentous fungus from human skin. Biocatalysis provides some advantages, such as high efficiency, high degrees of regioselectivity, chemoselectivity, and enantioselectivity. In order to optimize the small scale biocatalytic reaction of the (4S)-(+)-carvone by the filamentous fungus Phoma sp. was used headspace GC/MS methodology, factorial design of experiments and the response surface methodology (RSM) was performed using the biomass of the fungus, substrate mass and pH as parameters. It was observed that for all reactions conditions tested, forming the products (1?R,4S)-dihydrocarvone and (1S,4S)-dihydrocarvone. The most influential factor was pH, with the highest conversion rate (>95%) and diastereomeric excess (d.e.) (>80%) obtained at pH 5.0. Thus, it was demonstrated that human skin Phoma sp. fungus showed significant bioreduction activity and that headspace GC/MS is an efficient approach for real-time monitoring the biocatalysed reactions.