Improvement of the enantioselectivity and activity of lipase from Pseudomonas sp. via adsorption on a hydrophobic support: kinetic resolution of 2-octanol

Pseudomonas sp. lipase (PSL) was successfully immobilized on a novel hydrophobic polymer support through physical adsorption and the immobilized PSL was used for resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed from the immobilized PSL compared with free PSL. The effects of reaction conditions such as temperature, water activity, substrate molar ratio and the amount of immobilized lipase were investigated. Under optimum conditions, the residual (S)-2-octanol was recovered with 99.5% enantiomeric excess at 52.9% conversion. The results also indicated that the immobilized PSL could maintain 94% of its initial activity even after reusing it five times.     

A chemo-enzymatic process for sequential kinetic resolution of (R,S)-2-octanol under microwave irradiation

A combination of the enzymatic resolution and chemical racemization for the heterogeneous sequential kinetic resolution (SKR) was employed to resolve (R,S)-2-octanol under microwave irradiation. Mesoporous molecular sieves SBA-15, alumina and strong basic styrene anion exchange resin were screened and selected as the optimum supports to immobilize the lipase from Pseudomonas sp. (PSL), oxidant-Chromium trioxide (CrO₃) and reductant-Sodium borohydride (NaBH₄), respectively. The immobilized catalysts exhibited good reusability: it remained 90%, 72% and 80% of their initial activities after five reuses for the immobilized lipase, the immobilized oxidant and the immobilized reductant, respectively. Further, the E values of the immobilized PSL was increased from 23 under conventional heating to 40 under microwave irradiation in resolution of (R,S)-2-octanol. The immobilized catalysts were then used in SKR of (R,S)-2-octanol under microwave irradiation after optimizing the reaction media. Under the optimum conditions, (R)-2-octanol acetate was obtained at 99% enantiomeric excess with 84% yield in 2 h.     

Resolution of 2-octanol by SBA-15 immobilized Pseudomonas sp. lipase

Lipase from Pseudomonas sp. (PSL) was immobilized on SBA-15 (a highly ordered hexagonal array mesoporous silica molecular sieve) through physical adsorption and the immobilized PSL was used in resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed for the immobilized PSL compared with those of the free one. The effects of reaction conditions, such as solvents, temperature, water activity and substrate ratio were investigated. Under the optimum conditions, the residual (S)-2-octanol was recovered with 99% enantiomeric excess at 52% conversion. The results also indicated that the immobilized PSL maintained 90% of its initial activity even after reusing it five times.     

Immobilized Candida antarctica lipase B on ZnO nanowires/macroporous silica composites for catalyzing chiral resolution of (R,S)-2-octanol

ZnO nanowires were successfully introduced into a macroporous SiO₂ by in situ hydrothermal growth in 3D pores. The obtained composites were characterized by SEM and XRD, and used as supports to immobilize Candida antarctica lipase B (CALB) through adsorption. The high specific surface area (233 m²/g) and strong electrostatic interaction resulted that the average loading amount of the composite supports (196.8 mg/g) was 3–4 times of that of macroporous SiO₂ and approximate to that of a silica-based mesoporous material. Both adsorption capacity and the activity of the CALB immobilized on the composite supports almost kept unchanged as the samples were soaked in buffer solution for 48 h. The chiral resolution of 2-octanol was catalyzed by immobilized CALB. A maximum molar conversion of 49.1% was achieved with 99% enantiomeric excess of (R)-2-octanol acetate under the optimal condition: a reaction using 1.0 mol/L (R,S)-2-octanol, 2.0 mol/L vinyl acetate and 4.0 wt.% water content at 60 °C for 8 h. After fifteen recycles the immobilized lipase could retain 96.9% of relative activity and 93.8% of relative enantioselectivity.     

Microwave-assisted enzymatic resolution of (R,S)-2-octanol in ionic liquid

Combination use of microwave irradiation (MW) as heating mode and ionic liquid (IL) as reaction medium in enzymatic resolution of (R,S)-2-octanol with vinyl acetate as the acyl donor through transesterification by Novozym 435 was investigated. A synergistic effect of MW and IL [EMIM][NTf2], which was screened as the best reaction medium for this reaction, on improving enzyme activity and enantioselectivity was observed. The activity and enantioselectivity of Novozym 435 in [EMIM][NTf2] under MW were much higher than that in solvent free system under conventional heating, in solvent free system under MW, and in [EMIM][NTf2] under conventional heating, respectively. A systematic screening and optimization of the reaction parameters in [EMIM][NTf2] under MW were performed. Under the optimum conditions, 50% yield of (S)-2-octanol with 99% enantiomeric excess was obtained in 6 h. Furthermore, increased thermal stability and reusability of Novozym 435 under the combination use of MW and IL condition were also observed.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine by using microgel beads containing Pseudomonas cepacia lipase

Abstract

Pseudomonas cepacia lipase (PCL) was immobilized in alginate microgel beads by electrostatic dispersion. The high electrical potential applied in the immobilization process could significantly decrease the droplet size. The optimum conditions for lipase immobilization were 2% (w/v) alginate, 100 mM CaCl₂, 8 mg/mL enzyme, 4 kV electrical potential and 200 μm mean bead size. Under these conditions, 78.2 U/g of immobilized PCL activity was obtained with 39.1% retained activity and 57.2% immobilization efficiency. The immobilized PCL (PCL-CA) was subsequently used in the enantioselective hydrolysis of (R, S)-N-(2-ethyl-6-methylphenyl) alanine methyl ester. Although PCL-CA exhibited slightly lower activity than free PCL, it preserved the high enantioselectivity (E-value >?200), which afforded enantiomerically pure (R)-acid (99% e.e._p). Furthermore, PCL-CA exhibited higher thermal stability, storage and medium stability than that of free PCL. Batch-wise operational stability studies demonstrated that PCL-CA retained its initial activity for at least 10 cycles of hydrolysis.     

Stereoselective enzymatic esterification of 3-benzoylthio-2-methylpropanoic acid

Summary A key intermediate, S-(–)-3-benzoylthio-2-methylpropanoic acid (1) was made in high optical purity by the lipase-catalyzed stereoselective esterification of racemic 1 with methanol in an organic solvent system. Among various lipases evaluated, Amano P-30 lipase from Pseudomonas sp. efficiently catalyzed the esterification of 1 to yield R-(+) methyl ester and unreacted S-(–) 1. A reaction yield of 40 mol% and an optical purity of 97.2% were obtained for compound 1 at a substrate concentration of 0.1 m (22 mg/ml). Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (23 cycles) in the esterification reaction without loss of enzyme acitivity, productivity or optical purity. Among various solvents evaluated, toluene was found to be the most suitable organic solvent and methanol was the best alcohol for the esterification of racemic 1 by immobilized lipase. Substrate concentrations as high as 1.0 m were used in the esterification reaction. When the temperature was increased from 28° C to 60° C, the reaction time required for the esterification of 0.1 m substrate decreased from 16 h to 2 h. On increasing the methanol to substrate molar ratio from 1:1 to 4:1, the rate of esterification decreased. A lipase fermentation using Pseudomonas sp. ATCC 21 808 was developed. In the batch-fermentation process, 56 units/ml of extracellular lipase activity was obtained. A fed-batch process using soybean oil gave a significant increase in the lipase activity (126 units/ml). Crude lipase recovered from the filtrate by ethanol precipitation and immobilized on Accurel PP was also effective: S-(–) compound 1 was obtained in 35 mol% yield and 95% optical purity. Offsprint requests to: R. N. Patel     

Asymmetric reduction of 3-chloropropiophenone to (S)-3-chloro-1-phenylpropanol using immobilized Saccharomyces cerevisiae CGMCC 2266 cells

(S)-3-Chloro-1-phenylpropanol is an important chiral precursor for numerous antidepressants such as tomoxetine. A high enantiomeric excess (e.e.) of (S)-3-chloro-1-phenylpropanol can be achieved by asymmetric reduction of 3-chloropropiophenone using Saccharomyces cerevisiae CGMCC 2266 cells immobilized in calcium alginate. Thermal pretreatment of the immobilized cells at 50 °C for 30 min resulted in high enantioselectivity (99% e.e.) and good percent conversion (80%). The effects of various conditions on the reduction reaction were investigated. The optimal conditions were found to be as follows: sodium alginate concentration, 2%; bead diameter, 2 mm; temperature, 30 °C; re-culture time, 24 h; and batch addition of the substrate. After reusing these three times, the immobilized cells retained approximately 60% of their original catalytic activity with their enantioselectivity intact.     

Enantioselective enzymatic acetylation of racemic [4-[4α,6β (E)]]-6-[4,4-bis(4-fluorophenyl)-3-(1-methyl-1H-tetrazol-5-yl)-1,3-butadienyl]-tetrahydro-4-hydroxy-2H-pyran-2-one

Summary A chiral compound [4R-[4,6ß(E)]]-6-[4,4-bis(4-fluorophenyl)-3-(1-methyl-1H-tetrazol-5-yl)-1,3-butadienyl]-tetrahydro-4-hydroxy-2H-pyran-2-one (R-(+)-1) was prepared by the lipase-catalysed stereoselective acetylation of racemic 1 in an organic solvent. Chiral R-(+)-1 is a hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase inhibitor and a potential anticholesterol drug candidate. Among various lipases evaluated, lipase PS-30 from Pseudomonas species efficiently catalysed acetylation of the undesired enantiomer of racemic 1 to yield the S-(–)-acetylated product 2 and unreacted desired R-(+)-1. A reaction yield of 48 mol% and an optical purity of 98% were obtained for R-(+)-1 when the reaction was conducted in toluence as solvent in the presence of isopropenyl acetate as acyl donor. Lipase PS-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (five cycles) in the acetylation reaction without loss of enzyme activity, productivity, or optical purity of the R-(+)-1. The enzymatic acetylation process was scaled-up to 501 and a 640-l volume (preparative batches) at a substrate concentration of 4 g/l. R-(+)–1 was recovered from the preparative batches in 68–71% recovery yield with 98.5% gas chromatography homogeneity index and 98.5% optical purity. The S-(–) acetate 2 produced by the acetylation reaction was enzymatically hydrolysed by lipase PS-30 in a biphasic system to prepare the corresponding S-(–)-1.Correspondence to: R. N. Patel     

A Facile Method for Preparation of Polymerizable, Optically Active Ketoprofen Prodrug by Irreversible Lipase-catalysed Resolution

Summary An irreversible resolution of ketoprofen prodrug was developed by lipase-catalysed hydrolysis using corresponding vinyl ester as activated substrate in organic medium. The product obtained, (S)-ketoprofen vinyl ester would be used as a potential prodrug and a significant monomer for polymeric drug. Lipozyme^? immobilized from Mucor miehei showed the highest selectivity and activity after enzyme screening. The effect of solvent, water amount in the reaction medium and reaction temperature on the activity and enantioselectivity of Lipozyme^? was studied. Polymerizable, optically active ketoprofen prodrug could be obtained with excellent enantioselectivity (ee >99%, E ~ 400) in a mixture of dioxane/water (97.5/2.5, v/v) at 25 °C.     

Solvent effects on the enantioselectivity of the thermophilic lipase QLM in the resolution of (R,S)-2-octanol and (R,S)-2-pentanol

The thermophilic lipase QLM-catalyzed resolution of (R, S)-2-octanol and (R, S)-2-pentanol via transesterification was carried out in various organic solvents, and the solvent effects on the enzyme's enantioselectivity were investigated. A significant negative correlation between the enantiomeric ratio, E, and the size of the solvent molecules was observed. The highest E value, 21, was obtained in the small molecular-sized solvent dichloromethane when (R, S)-2-octanol was resolved with vinyl acetate as acyl donor. Thermodynamic analysis indicated that the difference in activation free energy between the two enantiomers was about 25.5% lower in dichloromethane than in the solvent-free system, and the change in the difference in activation entropy between the enantiomers was the main contributor to the changes in E values with the molecular size of solvents.     

Crosslinked aggregates of Rhizopus oryzae lipase as industrial biocatalysts: Preparation, optimization, characterization, and application for enantioselective resolution reactions

Lipase from Rhizopus oryzae (ROL) was immobilized as crosslinked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and simultaneous crosslinking with glutaraldehyde. The optimum conditions of the immobilization process were determined. Lipase CLEAs showed a twofold increase in activity when Tween 80‐pretreated lipase was used for CLEA preparation. CLEAs were shown to have several advantages compared to free lipase. CLEAs were more stable at 50°C and 60°C as well as for a wide range of pH. After incubation at 50°C, CLEA showed 74% of initial activity whereas free enzyme was totally inactivated. Reduction of Schiff bases has been performed for the first time in the CLEA preparation process significantly improving the chemically modified CLEAs' reusability, thus providing an enzyme with high potential for recycling even under aqueous reaction conditions where enzyme leakage is, in general, one of the major problems. The CLEA retained 91% activity after 10 cycles in aqueous medium. The immobilized enzyme was used for kinetic resolution reactions. Results showed that immobilization had an enhancing effect on the conversion (c) as well as on the enantiomeric ratio (E). ROL CLEA displayed five times higher enantioselectivity for the hydrolysis of (R,S)‐1‐phenylethyl acetate and likewise 1.5 times higher enantioselectivity for the transesterification of racemic (R–S)‐1‐phenylethanol with vinylacetate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 937–945, 2012 This article was published online on June 26, 2012. An edit was subsequently requested. This notice is included in the online and print versions to indicate that both have been corrected [27 June 2012].     

Asymmetric Hydrolysis of Racemic 2-Oxazolidinone Esters with Lipases

The enzymatic hydrolysis of (R, S)-5-acyloxymethyl-3-alkyl-oxazolidin-2-one I and the behavior of (S)-I for extraction with an organic solvent were examined so as to extend the biological resolution to racemates, and to learn about more appropriate combinations of substrates with lipases on the asymmetric hydrolysis. The combination of (R, S)-5-hexanoyloxymethyl-3-tert-butyl-oxazolidin-2-one 4 with lipoprotein lipase Amano 3 (L. P. L. Amano 3, origin; Pseudomonas aeruginosa) and that of (R, S)-5-octanoyloxymethyl-3-isopropyl-oxazolidin-2-one 14 with L. P. L. Amano 3 efficiently gave (S)-5-hydroxymethyl-3-tert-butyl-oxazolidin-2-one (S)-lla (99% e.e.) and (S)-5-hydroxymethyl-3-isopropyl-oxazolidin-2-one (S)-IIb (99% e.e.),respectively. (S)-IIa and (S)-IIb could be considered to be favorable intermediates for preparing optically active β-blockers.     

Immobilization of lipase by salts and the transesterification activity in hexane

Lipases from six different sources were immobilized on Celite and five types of salt. The transesterification activities in hexane for lipases immobilized on EDTA-Na₂ increased by 463% for the lipase from Candida rugosa (CRL), 2700% for the lipase from Candida sp. (CSL) and 1215% for the lipase from Pseudomonas sp. (PSL), compared to the salt-free enzyme. With 0.5% sucrose for CRL or 1% sorbitol for PSL as the lyoprotectant during lyophilization process, transesterification activity increased by 100% and 13%, respectively, compared to the immobilized enzyme on EDTA-Na₂ without lyoprotectant.     

Enantioselective Hydrolysis of α-Cyano-3-phenoxybenzyl Acetate with Arthvobacter Lipase

Lipase-catalyzed enantioselective hydrolysis of the acetic ester of racemic α-cyano-3-phenoxybenzyl alcohol (CPBA) was examined to prepare (S)-CPBA. Most of the lipases tested hydrolyzed (S)-CPBA acetate preferentially, while Candida cylindracea lipase favored (R)-CPBA acetate. Enantioselective hydrolysis by Arthrobacter lipase gave the optically pure (S)-CPBA in the reaction mixture of pH 4.0. The kinetic studies showed that (R)-CPBA acetate reacted as a competitive inhibitor. The Arthrobacter lipase solution in the water/oil biphasic reaction system could be used repeatedly. The lipase immobilized to resins had insufficient activity or low operational stability for the repeated batch reaction. The unhydrolyzed (R)-CPBA acetate was racemized by heating with triethylamine and could be reused as the substrate of the enzymatic hydrolysis. A chemico-enzymatic process for the preparation of (S)-CPBA was developed based on these studies.     

Chemoenzymatic resolution of racemic Wieland–Miescher and Hajos–Parrish ketones

Enantiospecific microbial reduction of bicyclic ketones was described. Racemic Wieland–Miescher (1) and Hajos–Parrish (2) ketones were used as substrates. In a 4-h biotransformation of Hajos–Parrish ketone (2) using the strain of Didymosphaeria igniaria an optically pure ketone (R)-2 was obtained, whereas the (S)-2 ketone underwent reduction to (4aS,5S)-4 alcohol with 100% of enantiomeric excess and with over 60% of diastereoisomeric excess. Jones oxidation of the alcohol obtained in the biotransformation gave an optically pure ketone (S)-2. Enzymatic system of Coryneum betulinum reduced the (R)-2 ketone to (4aR,5S)-4 alcohol with a high enantiomerical purity in a 6-day reaction. Wieland-Miescher (1) ketone was transformed by these microorganisms in an analogous way, but the reaction times were longer.     

Kinetic resolution of α-lipoic acid via enzymatic differentiation of a remote stereocenter

Kinetic resolution of α-lipoic acid, a case of remote stereocenter discrimination, was accomplished using lipase from Aspergillus oryzae WZ007. Performance of this lipase was investigated for enantioselective esterification of (S)-α-lipoic acid, leaving the target product (R)-α-lipoic acid in unreacted form. The effects of chain length of alcohol, type of solvent, molar ratio of alcohol:acid, and reaction temperature were studied. The optimum reaction conditions were found to be esterification with n-octanol at 50°C in heptane with an alcohol:acid molar ratio of 5:1. The conversion rate of α-lipoic acid was 75.2%, with an enantiomeric excess of 92.5% towards unreacted substrate in a reaction time of 48 h.     

Synthesis of the Deuterated Sex Pheromone Components of the Grape Borer,Xylotrechus pyrrhoderus

Adult males of the grape borer, Xylotrechus pyrrhoderus, secrete (S)-2-hydroxy-3-octanone [(S)-1] and (2S,3S)-2,3-octanediol [(2S,3S)-2] from their nota of prothoraces as sex pheromone components. Their structural similarity suggests that one of them is the biosynthetic precursor of the other component. In order to confirm the biochemical conversion, deuterated derivatives of both components were synthesized by starting from a Wittig reaction between hexanal and an ylide derived from D₅-iodoethane and ending with enantiomeric resolution by chiral HPLC. The molecular ions of 1 and 2 could scarcely be detected by using a GC-MS analysis, and the labeled compounds showed similar mass spectra to the unlabeled pheromone components. However, several fragment ions, including four deuterium atoms, were observed in the mass spectra of their acetate derivatives, indicating that the conversion could be confirmed by examining a compound with the diagnostic ions after acetylation of the volatiles collected from insects treated with the labeled precursors.     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP?g^?1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^?1 µg^?1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Synthesis and Antimicrobial Activity of Chiral cis-Cycloheximide Isomers

Such (+)- and (?)-cis-cycloheximide isomers as isocyclohcximide (1a, 1b), α-epiisocycloheximide (2a, 2b) and neocycloheximide (3a, 3b) were synthesized by aldol condensation of (?)-(2R, 4R)- and (+)-(2S, 4S)-cis-2,4-dimethyl-1-cyclohexanone (5a, 5b). obtained by microbial resolution, with 4-(2-oxoethyl)-2,6-piperidinedione (7). The absolute configuration of the (?)-cis-ketone 5a was confirmed by chemical correlation with natural (2S, 4S, 6S, αR)-cycloheximide (4). The newly synthesized isomer, (?)-α-epiisocycloheximide (2b), showed strong antimicrobial activity against S. cerevisiae andP. oryzae close to that of natural cycloheximide (4).