Enantioselectivity of Epoxide Formation from Halohydrins by Means of Flavobacterium Rigense

The formation of epoxides from several halohydrins was achieved using resting cells from Flavobacterium rigense. The reaction showed a high substrate specificity for halohydrins with a terminal halogen atom but only low enantioselectivity (12–58% e.e.). The epoxides always had the (S)-configuration. Substrates which in the halogen atom was replaced by another leaving group (-O-SO₂CH₃, -O-Tos, -N₃) were not accepted. An attempt to improve the enantioselectivity by using a two phase system consisting of an aqueous and an organic solvent phase was not successful.     

Asymmetric bioreduction of p-haloacetophenones by Mucor

Abstract

A number of p-haloacetophenones were asymmetrically bioreduced to their corresponding (S)-alcohols by Mucor sp. CG10 with good conversion and excellent enantioselectivity. The results showed that the electronic effects of the halogen substituent (X-group) affected the conversion of the substrates and the enantioselectivity of the reaction. The trend observed was as the X-group at the para-position became more electron donating from F, to Cl, Br and I, the conversion of substrates decreased, while the enantioselectivity increased.     

Microbial reduction of ethyl acetoacetate to ethyl (R)-3-hydroxybutyrate in an ionic liquid containing system

A hydrophilic ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF₄) was successfully employed as co-solvent for asymmetric bioreduction of ethyl acetoacetate (EOB) to ethyl (R)-3-hydroxybutyrate (R-EHB) catalyzed by Pichia membranaefaciens Hansen ZJPH07 cells. The results demonstrated that the addition of [BMIM]BF₄ in reaction system can markedly reduce the substrate inhibition and moderately improve the enantioselectivity compared to that in monophasic aqueous system. Among different alcohols and carbohydrates tried as co-substrate, glucose was a proper electron donor. Although isopropanol gave the best enantioselectivity with the highest yield, S-enantiomer was obtained. To optimize the bioreduction, some reaction parameters for the biosynthesis of R-EHB in this IL-containing system were investigated, such as temperature, buffer pH, shaking speed, substrate concentration, wet cells concentration and reaction time. Under the optimum conditions, best conversion of 77.8% and product enantiomeric excess (e.e.) of 73.0% were obtained. A comparative study was performed either in the presence or in the absence of [BMIM]BF₄, higher reaction yield (77.8% versus 68.5%) and product e.e. (73.0% versus 65.1%) were observed in IL-containing system with 0.55 M of the substrate, but 0.35 M of substrate concentration for the reduction in aqueous system without the addition of [BMIM]BF₄.     

Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production

Candida rugosa lipase (CRL) was immobilized on Amberlite XAD 7 and the advantage of immobilization under the best reaction conditions in achieving high activity and enantioselectivity was shown for the hydrolysis of racemic Naproxen methyl ester. The performance of CRL was found to be better when the enzyme was immobilized at the temperature and pH values where higher conversion and enantioselectivity were obtained. The effects of immobilized lipase load, temperature, pH and substrate concentration on the conversion and enantioselectivity toward S-Naproxen production in aqueous phase/isooctane biphasic batch system were also evaluated. The increase in immobilized lipase load in 320–800 U/mL range increased the conversion of the substrate and enantioselectivity for S-Naproxen. The kinetic resolution of racemic Naproxen methyl ester conducted at the temperatures of 40, 45 and 50 °C and at the pH values of 4, 6, 7.5 and 9 resulted in the highest conversion and enantioselectivity at 45 °C and pH 6. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. CRL, which was immobilized at the temperature and pH values where the enzyme was more enantioselective, was successfully used in three successive batch runs each of 180 h. The highest enantiomeric ratio achieved in the S-Naproxen production was 174.2 with the conversion of 49%.     

Enantioselective transesterification of racemic phenyl ethanol and its derivatives in organic solvent and ionic liquid using Pseudomonas aeruginosa lipase

The transesterification of 1-phenyl ethanol has been carried out using lipases from Pseudomonas aeruginosa MTCC 5113, to obtain chirally pure aryl ethanols with good yield and excellent enantioselectivity. The lipase from P. aeruginosa gave good conversion and moderate enantioselectivity (ee) in organic solvents, however, when the catalytic amount of ionic liquids were added in the reaction mixture, excellent enantioselectivity was obtained. Moreover, the change in enantiomer preference was seen in the presence of catalytic amount of ionic liquids. The findings revealed that hydrophobic ionic liquids (two-phase system) were the best solvents and 4-substituted aryl ethanols were the pre-eminent substrates for such type of reactions. The preparative scale (5 g) transesterification of 1-phenylethanol using lipases from P. aeruginosa yielded S-(−)-1-phenyl ethanol with 39% yield and >99% ee in hexane and 46% yield and >99% ee in [BMIm][PF₆].     

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).     

Effect of ionic liquid [BMIM][PF<Subscript>6</Subscript>] on asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The effect of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF₆]) on the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) to synthesize optical active ethyl 2-hydroxy-4-phenylbutyrate (EHPB) catalyzed by Saccharomyces cerevisiae was investigated. (R)-EHPB [70.4%, e.e.(R)] is obtained using ethyl ether or benzene as the solvent. The main product is (S)-EHPB [27.7%, e.e.(S)] in [BMIM][PF₆]. However, in ionic liquid-water (10:1, v/v) biphasic system, the enantioselectivity of the reduction is shifted towards (R)-side, and e.e.(R) is increased from 6.6 to 82.5% with the addition of ethanol (1%, v/v). The effect of the use of [BMIM][PF₆] as an additive in relatively small amounts on the reduction was also studied. We find that there is a decline in the enantioselectivity of the reduction in benzene. In addition, a decrease in the conversion of EOPB and the yield of EHPB with increasing [BMIM][PF₆] concentrations occurs in either organic solvent–water biphasic systems or benzene.     

Enantioselective reduction of 4-chromanone and its derivatives by selected filamentous fungi

Biotransformation of 4-chromanone and its derivatives in the cultures of three biocatalysts: Didymosphaeria igniaria, Coryneum betulinum and Chaetomium sp. is presented. The biocatalysts were chosen due to their capability of enantiospecific reduction of low-molecular-weight ketones (acetophenone and its derivatives and α- and β-tetralone). The substrates were reduced to the respective S-alcohols with high enantiomeric excesses, according to the Prelog's rule. In the culture of Chaetomium sp. after longer biotransformation time an inversion of configuration of the formed alcohols was also observed. The highest yield of transformation was observed for 6-methyl-4-chromanone. In all the tested cultures, the higher was the molecular weight of a chromanone, the lower conversion percent was observed.     

Chemoenzymatic Preparation of Optically Active Long-Chain 3-Hydroxyalkanoates

The synthesis of optically active 3-hydroxyalkanoates of varying chain length (C_lo, C₁₄ and C₁₈) was investigated using chemoenzymatic methods. While bakers' yeast mediated reduction of 3-oxoalkanoates gave (R)-3-hydroxyalkanoates (e.e. >80%, yield —10%), both enantiomers were obtained by enzymatic resolution of racemic methyl 3-butanoyloxyalkanoates using Geotrichum candidum lipase with moderate to good optical purity (e.e. 32–92%, yield 40–60% for both enantiomers). Lipase-catalysed interesterification, however, was found to proceed with similar enantioselectivity but with slow rate of conversion.     

Immobilization of Candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester

Candida rugosa lipase (CRL) was immobilized on glutaraldehyde-activated aminopropyl glass beads by using covalent binding method or sol-gel encapsulation procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of RSi(OCH₃)₃ and Si(OCH₃)₄. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP). It has been observed that the percent activity yield of the encapsulated lipase was 166.9, which is 5.5 times higher than that of the covalently immobilized lipase. The enantioselective hydrolysis of racemic Naproxen methyl ester by immobilized lipase was studied in aqueous buffer solution/isooctane reaction system and it was noticed that particularly, the glass beads based encapsulated lipases had higher conversion and enantioselectivity compared to covalently immobilized lipase. In short, the study confirms an excellent enantioselectivity (E > 400) for the encapsulated lipase with an ee value of 98% for S-Naproxen.     

Enantioselective esterification of ibuprofen in supercritical carbon dioxide by immobilized lipase

The enantioselective esterification of racemic ibuprofen with n-propanol by immobilized Mucor miehel lipase in supercritical carbon dioxide was studied. The enantiomeric excess of the product (ee_p) was 70 % at 15...20 % conversion. The enantioselectivity was faintly affected by temperature and the concentration of ibuprofen and lipase. The optimum temperature was 45 °C. The initial reaction rate increased with pressure, but enantioselectivity was not affected by pressure changes. The reaction rates in supercritical carbon dioxide at optimized conditions and in n-hexane were similar.     

Combinatorial assembly platform enabling engineering of genetically stable metabolic pathways in cyanobacteria

Cyanobacteria are simple, efficient, genetically-tractable photosynthetic microorganisms which in principle represent ideal biocatalysts for CO₂ capture and conversion. However, in practice, genetic instability and low productivity are key, linked problems in engineered cyanobacteria. We took a massively parallel approach, generating and characterising libraries of synthetic promoters and RBSs for the cyanobacterium Synechocystis sp. PCC 6803, and assembling a sparse combinatorial library of millions of metabolic pathway-encoding construct variants. Genetic instability was observed for some variants, which is expected when variants cause metabolic burden. Surprisingly however, in a single combinatorial round without iterative optimisation, 80% of variants chosen at random and cultured photoautotrophically over many generations accumulated the target terpenoid lycopene from atmospheric CO₂, apparently overcoming genetic instability. This large-scale parallel metabolic engineering of cyanobacteria provides a new platform for development of genetically stable cyanobacterial biocatalysts for sustainable light-driven production of valuable products directly from CO₂, avoiding fossil carbon or competition with food production.     

Layer-by-Layer coated tyrosinase: An efficient and selective synthesis of catechols

Agaricus bisporous tyrosinase was immobilized on commercial available epoxy-resin Eupergit®C250L and then coated by the Layer-by-Layer method (LbL). The two novel heterogeneous biocatalysts were characterized for their morphology, pH and storage stability, kinetic properties (K_m, V_max, V_max/K_m) and reusability. These biocatalysts were used for the efficient and selective synthesis of bioactive catechols under mild and environmental friendly experimental conditions. Ascorbic acid was added in the reaction medium to inhibit the formation of ortho-quinones, thus avoiding the known enzyme suicide inactivation process. Catechols were obtained mostly in quantitative yields and conversion of substrate. Tyrosinase immobilized on Eupergit®C250L and coated by the LbL method showed better catalytic activities, higher pH and storage stability, and reusability with respect to immobilized uncoated tyrosinase. Since chemical procedures to synthesize catechols are often expensive and with high environmental impact, the use of immobilized tyrosinase represents an efficient alternative for the preparation of this family of bioactive compounds.     

Towards catalyst compartimentation in combined chemo- and biocatalytic processes: Immobilization of alcohol dehydrogenases for the diastereoselective reduction of a β-hydroxy ketone obtained from an organocatalytic aldol reaction

The alcohol dehydrogenases (ADHs) from Lactobacillus kefir and Rhodococcus sp., which earlier turned out to be suitable for a chemoenzymatic one-pot synthesis with organocatalysts, were immobilized with their cofactors on a commercially available superabsorber based on a literature known protocol. The use of the immobilized ADH from L. kefir in the reduction of acetophenone as a model substrate led to high conversion (>95%) in the first reaction cycle, followed by a slight decrease of conversion in the second reaction cycle. A comparable result was obtained when no cofactor was added although a water rich reaction media was used. The immobilized ADHs also turned out to be suitable catalysts for the diastereoselective reduction of an organocatalytically prepared enantiomerically enriched aldol adduct, leading to high conversion, diastereomeric ratio and enantioselectivity for the resulting 1,3-diols. However, at a lower catalyst and cofactor amount being still sufficient for biotransformations with “free” enzymes the immobilized ADH only showed high conversion and >99% ee for the first reaction cycle whereas a strong decrease of conversion was observed already in the second reaction cycle, thus indicating a significant leaching effect of catalyst and/or cofactor.     

Identification and characterization of a highly S-enantioselective halohydrin dehalogenase from Tsukamurella sp. 1534 for kinetic resolution of halohydrins

Halohydrin dehalogenases are remarkable enzymes which possess promiscuous catalytic activity and serve as potential biocatalysts for the synthesis of chiral halohydrins, epoxides and β-substituted alcohols. The enzyme HheC exhibits a highly R enantioselectivity in the processes of dehalogenation of vicinal halohydrins and ring-opening of epoxides, which attracts more attentions in organic synthesis. Recently dozens of novel potential halohydrin dehalogenases have been identified by gene mining, however, most of the characterized enzymes showed low stereoselectivity. In this study, a novel halohydrin dehalogenase of HheA10 from Tsukamurella sp. 1534 has been heterologously expressed, purified and characterized. Substrate spectrum and kinetic resolution studies indicated the HheA10 was a highly S enantioselective enzyme toward several halohydrins, which produced the corresponding epoxides with the ee (enantiomeric excess) and E values up to >99% and >200 respectively. Our results revealed the HheA10 was a promising biocatalyst for the synthesis of enantiopure aromatic halohydrins and epoxides via enzymatic kinetic resolution of racemic halohydrins. What’s more important, the HheA10 as the first individual halohydrin dehalogenase with the highly S enantioselectivity provides a complementary enantioselectivity to the HheC.     

Lipases fromRhizomucor miehei andHumicola lanuginosa: Modification of the lid covering the active site alters enantioselectivity

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:E _S =8.5;H. lanuginosa:E _S =10.5), but theR-enantiomer of phenyl 2-methyldecanoate (E _R =2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (E _R =2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (E _S =2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (E _S =1.9) and increased the enantioselectivity with the aromatic ester (E _R =4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (E _S =17.4) and a decreased enantioselectivity with the phenyl ester (E _R =2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.     

Immobilized Baliospermum montanum hydroxynitrile lyase catalyzed synthesis of chiral cyanohydrins

Hydroxynitrile lyase (HNL) catalyzed enantioselective CC bond formation is an efficient approach to synthesize chiral cyanohydrins which are important building blocks in the synthesis of a number of fine chemicals, agrochemicals and pharmaceuticals. Immobilization of HNL is known to provide robustness, reusability and in some cases also enhances activity and selectivity.We optimized the preparation of immobilization of Baliospermium montanum HNL (BmHNL) by cross linking enzyme aggregate (CLEA) method and characterized it by SEM. Optimization of biocatalytic parameters was performed to obtain highest % conversion and ee of (S)-mandelonitrile from benzaldehyde using CLEA-BmHNL. The optimized reaction parameters were: 20 min of reaction time, 7 U of CLEA-BmHNL, 1.2 mM substrate, and 300 mM citrate buffer pH 4.2, that synthesized (S)-mandelonitrile in ∼99% ee and ∼60% conversion. Addition of organic solvent in CLEA-BmHNL biocatalysis did not improve in % ee or conversion of product unlike other CLEA-HNLs. CLEA-BmHNL could be successfully reused for eight consecutive cycles without loss of conversion or product formation and five cycles with a little loss in enantioselectivity. Eleven different chiral cyanohydrins were synthesized under optimal biocatalytic conditions in up to 99% ee and 59% conversion, however the % conversion and ee varied for different products. CLEA-BmHNL has improved the enantioselectivity of (S)-mandelonitrile synthesis compared to the use of purified BmHNL. Nine aldehydes not tested earlier with BmHNL were converted into their corresponding (S)-cyanohydrins for the first time using CLEA-BmHNL. Among the eleven (S)-cyanohydrins syntheses reported here, eight of them have not been synthesized by any CLEA-HNL. Overall, this study showed preparation, characterization of a stable, robust and recyclable biocatalyst i.e. CLEA-BmHNL and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins.     

Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2-O-Isopropylideneglycerol Esters

Escherichia coli has been widely used as an expression host for the identification of desired biocatalysts through screening or selection assays. We have previously used E. coli in growth selection and screening assays for identification of Bacillus subtilis lipase variants (located in the periplasm) with improved activity and enantioselectivity toward 1,2-O-isopropylideneglycerol (IPG) esters. In the course of these studies, we discovered that E. coli itself exhibits significant cytoplasmic esterase activity toward IPG esters. In order to identify the enzyme (or enzymes) responsible for this esterase activity, we analyzed eight E. coli knockout strains, in which single esterase genes were deleted, for their ability to hydrolyze IPG butyrate. This approach led to the identification of esterase YbfF as the major E. coli enzyme responsible for the hydrolytic activity toward IPG esters. The gene coding for YbfF was cloned and overexpressed in E. coli, and the corresponding protein was purified and characterized for its biocatalytic performance. YbfF displays a high level of activity toward IPG butyrate and IPG caprylate and prefers the R-enantiomer of these substrates, producing the S-enantiomer of the IPG product with high enantiomeric excess (72 to 94% ee). The enantioselectivity of YbfF for IPG caprylate (E = 40) could be significantly enhanced when using dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as cosolvents in kinetic resolution experiments. The enzyme also shows high enantioselectivity toward 1-phenylethyl acetate (E ≥ 200), giving the chiral product (R)-1-phenylethanol with >99% ee. The high activity and enantioselectivity of YbfF make it an attractive enzyme for organic synthesis.     

Completing the series of BVMOs involved in camphor metabolism of Pseudomonas putida NCIMB 10007 by identification of the two missing genes, their functional expression in E. coli, and biochemical characterization

The camphor-degrading Baeyer?CVilliger monooxygenases (BVMOs) from Pseudomonas putida NCIMB 10007 have been of interest for over 40?years. So far the FMN- and NADH-dependent type II BVMO 3,6-diketocamphane 1,6-monooxygenase (3,6-DKCMO) and the FAD- and NADPH-dependent type I BVMO 2-oxo-?³-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) have not been entirely studied, since it was not possible to produce those enzymes in satisfactory amounts and purity. In this study, we were able to clone and recombinantly express both enzymes and subsequently use them as biocatalysts for various mono- and bicyclic ketones. Full conversion could be reached with both enzymes towards (±)-cis-bicyclo[3.2.0]hept-2-en-6-one and with 3,6-DKCMO towards (?)-camphor. Further OTEMO gave full conversion with norcamphor. OTEMO was found to have a pH optimum of 9 and a temperature optimum of 20?°C and converted (±)-cis-bicyclo[3.2.0]hept-2-en-6-one with a k _cat/K _M value of 49.3?mM^?1?s^?1.     

Scale-up of Baeyer–Villiger monooxygenase-catalyzed synthesis of enantiopure compounds

Several Baeyer–Villiger monooxygenases converting a wide spectrum of substrates have been discovered, cloned, and characterized throughout the last few years. Still, only a few of them are applicable for large-scale conversion predominantly due to their sensitivity towards high substrate and/or product concentrations. The recently cloned and characterized 4-hydroxyacetophenone monooxygenase from Pseudomonas putida JD1 shows excellent enantioselectivity towards 3-phenyl-2-butanone with E?>?100 but is inhibited by concentrations >10 mM of both substrate and product. This obstacle could be circumvented by in situ substrate feed and product removal using a hydrophobic Lewatit® adsorbent resin. Thus, the concentration of 3-phenyl-2-butanone could be increased from 1.4 to >26 mM without significant reduction in conversion.