Resolution of 2-octanol via immobilized Pseudomonas sp. lipase in organic medium

Abstract

Pseudomonas sp. lipase (PSL) immobilization was performed using three different protocols. Lipase immobilized on Diaion HP20 (HP20-PSL) exhibited the highest catalytic activity and stability in the kinetic resolution of racemic 2-octanol. The reaction rate of HP20-PSL was approximately 20 times higher than that of free PSL and the residual activities of HP20-PSL and free PSL were respectively 84% and 19% after incubation in the reaction medium for 72 h. A study of the effect of different reaction parameters on HP20-PSL-catalyzed resolution of (R,S)-2octanol showed that the optimal water content of the immobilized matrix and the optimal molar ratio of vinyl acetate to 2-octanol were 60 ± 5% and 2.5:1, respectively. Under the optimized reaction conditions, (S)-2-octanol of high optically purity (enantiomeric excess > 99%) could be recovered at 53% conversion rate, and HP20-PSL could be reused for ten cycles without significant decrease in its activity and enantioselectivity.     

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.     

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.     

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.     

Enhancement of the activity and enantioselectivity of lipase in organic systems by immobilization onto low-cost support

Lipase from Arthrobacter sp. was immobilized onto low-cost diatomite materials using different protocols for the resolution of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one (HMPC) by asymmetric acylation. The support surface was grafted various functional groups including methacryloxypropyl, vinyl, octyl, dodecyl and γ-(aminopropyl)-glutaraldehyde. These modifications resulted in various mechanisms during the immobilization and thus introduced different characteristics to the prepared lipases. The interfacially adsorbed lipase onto dodecyl-modified support exhibited both higher activity and stability among these immobilized preparations. The modified enzyme-aggregate coating method was performed based on interfacial adsorption in our work, and the characteristics of this immobilized lipase were investigated and compared with those by cross-linking and interfacial adsorption methods. It was shown that the enzyme-aggregate coated lipase yielded the highest activity with a recovered activity of 8.5-fold of the free enzyme, and the highest operational stability with 85% of initial activity remained after 10 recycles. Excellent enantioselectivity (E ≥ 400, with e.e. = 99% of S-HMPC) was obtained for most lipase preparations in our paper (E = 85 for the free enzyme).     

复合诱变选育高产脂肪酶黑曲霉菌株及其固定化酶性质

通过硫酸二乙酯(DES)和微波复合诱变,获得遗传性状稳定的高产脂肪酶黑曲霉突变菌株CM2,酶活达174.93 U/mL.对菌株CM2培养条件的优化,以橄榄油和(NH_4)_2SO_4为最佳碳、氮源,在28℃、pH 7.5的条件下,发酵CM2菌株68 h,脂肪酶活为180.52 U/mL.大孔树脂固定化脂肪酶在35～55℃和pH 7.5～9.5之间有很好的稳定性.游离酶和固定化酶的表观失活活化能分别为52.6842 kJ/mol和30.8391 kJ/mol,固定化酶对温度的敏感度降低,耐受性增强.在微水相中脂肪酶催化2-辛醇和乙酸乙烯酯不对称酯交换反应中,(S)-乙酸辛酯的对映选择性高(游离酶e.e.s 85.7%;固定化酶e.e.s 87.7%),显示了该固定化酶在2-辛醇的手性拆分方面具有良好的应用前景.     

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     

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.     

Kinetic resolution of rac-alkyl alcohols via lipase-catalyzed enantioselective acylation using succinic anhydride as acylating agent

With succinic anhydride as acylating agent, three commercial lipases – Candida antarctica lipase B (CALB), Pseudomonas cepacia lipase and Pseudomonas fluorescens lipase – were employed in the kinetic resolution of a series of rac-alkyl alcohols: 2-butanol, 2-pentanol, 2-hexanol, 2-heptanol, 2-octanol, 3-hexanol, 3-methyl-2-butanol, 6-methyl-5-heptene-2-ol, 3-methyl-2-cyclohexene-1-ol and 2-methyl-1-pentanol. The most effective tested enzyme, immobilized CALB, was able to resolve most of the alcohols with high enantioselectivity, even higher (with enantiomeric ratios up to 115 and 91, for 3-hexanol and 3-methyl-2-butanol, respectively) than when vinyl acetate was used as the acylating agent. More importantly, the unreacted alcohol and the monoester succinate produced could be easily separated by a simple aqueous base-organic solvent liquid–liquid extraction. Using succinic anhydride as acylating agent and CALB, enantiomerically pure (S)-2-pentanol with 99% ee and (R)-2-pentanol with 95% ee were prepared in gram-scale reactions.     

Immobilized Pseudomonas sp. lipase: A powerful biocatalyst for asymmetric acylation of (±)-2-amino-1-phenylethanols with vinyl acetate

Pseudomonas sp. lipase was immobilized onto glutaraldehyde-activated Florisil^® support via Schiff base formation and stabilized by reducing Schiff base with sodium cyanoborohydride. The immobilization performance was evaluated in terms of bound protein per gram of support (%) and recovered activity (%). A 4-factor and 3-level Box–Behnken design was applied for the acylation of (±)-2-(propylamino)-1-phenylethanol, a model substrate, with vinyl acetate and the asymmetric acylations of other (±)-2-amino-1-phenylethanols with different alkyl substituents onto nitrogen atom such as (±)-2-(methylamino)-1-phenylethanol, (±)-2-(ethylamino)-1-phenylethanol, (±)-2-(butylamino)-1-phenylethanol and (±)-2-(hexylamino)-1-phenylethanol were performed under the optimized conditions. The optimal conditions were bulk water content of 1.8%, reaction temperature of 51.5 °C, initial molar ratio of vinyl acetate to amino alcohol of 1.92, and immobilized lipase loading of 47 mg mL^?1. (R)-enantiomers of tested amino alcohols were preferentially acylated and the reaction purely took place on the hydroxyl group of 2-amino-1-phenylethanols. The increase of alkyl chain length substituted onto nitrogen atom caused an increase in the acylation yield and ee values of (S)-enantiomers. Enantiomeric ratio values were >200 for all the reactions. Our results demonstrate that the immobilized lipase is a promising biocatalyst for the preparation of (S)-2-amino-1-phenylethanols and their corresponding (R)-esters via O-selective acylation of (±)-2-amino-1-phenylethanols with vinyl acetate.     

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].     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

Enantioselective enzymatic hydrolysis of racemic drugs by encapsulation in sol–gel magnetic sporopollenin

Candida rugosa lipase was encapsulated within a sol–gel procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of octyltriethoxysilane and tetraethoxysilane in the presence of magnetic sporopollenin. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e., the hydrolysis of p-nitrophenylpalmitate (p-NPP), and the enantioselective hydrolysis of racemic naproxen methyl ester, mandelic acid methyl ester or 2-phenoxypropionic acid methyl ester that were studied in aqueous buffer solution/isooctane reaction system. The encapsulated magnetic sporopollenin (Spo-M-E) was found to give 319 U/g of support with 342% activity yield. It has been observed that the percent activity yields and enantioselectivity of the magnetic sporopollenin encapsulated lipase were higher than that of the encapsulated lipase without support. The substrate specificity of the encapsulated lipase revealed more efficient hydrolysis of the racemic naproxen methyl ester and 2-phenoxypropionic acid methyl ester than racemic mandelic acid methyl ester. It was observed that excellent enantioselectivity (E > 400) was obtained for encapsulated lipase with magnetic sporopollenin with an ee value of S-Naproxen and R-2 phenoxypropionic acid about 98%.     

Immobilized copper-ion affinity adsorbent based on a cross-linked β-cyclodextrin polymer for adsorption of Candida rugosa lipase

Lipase from Candida rugosa was immobilized on a β-cyclodextrin-based polymer by adsorption and subsequent cross-linking with epichlorohydrin (EP-CD). The ligand iminodiacetic acid (IDA) was then bonded with the cross-linked β-cyclodextrin (EP-CD-IDA). This affinity adsorbent was further chelated with Cu^{2 +} for the purpose of binding affinity and stability. The properties of the immobilized lipase were assayed and compared with those of the free enzyme. Results showed that 266 µg protein with an activity of 17.85 U was bound per gram of matrix, giving 188% of the specific activity of the free enzyme and a total recovered activity of 79.7% under the optimum conditions. The pH and thermal stabilities of lipase were improved after immobilization on the β-cyclodextrin-based polymer (EP-CD-IDA-Cu^{2 +}). In addition, experimental results indicated that the residual activity of the immobilized lipase was 50% after eight cycles of reuse.     

Comparison of differently modified Pseudomonascepacia lipases in enantioselective preparation of a chiral alcohol for agrochemical use

Three methods for enzyme modification/immobilization were compared to enhance the catalytic performance of a commercially available lipase, Lipase PS from Pseudomonascepacia, in highly enantioselective transesterification of an agrochemically useful sec-alcohol, (R,?S)-HMPC [=(R,?S)-4-hydroxy-3-methyl-2-(2′-propenyl)-2-cyclopenten-1-one], with vinyl acetate as both acyl donor and reaction medium. The stearic acid-coated lipase showed the highest catalytic activity, with a specific activity improved by 54 times over the native lipase. The microcrystal salt-supported lipase and celite-adsorbed lipase also displayed much better performance as compared with the native lipase. All the three modified lipase preparations showed a similar thermal stability to that of the native enzyme. The enantioselectivity (E-value) was also quite satisfactory in all the cases (E>100 at 30°C), though a trend of slight decline was also observed with the temperature increase in the range of 25–60°C. The optimum aqueous pH, from which the modified lipases were prepared, was 6.0–7.0. A low water activity (a_w) of ca. 0.1 was favorable for all the three modified lipases. The stearic acid-coated lipase displayed prominent advantages in catalyzing the transesterification reaction at a very high (R,?S)-HMPC concentration up to 1.0?M.     

Experiment and simulation on kinetic resolution of (R,S)-2-chloromandelic acid by enzymatic transesterification

Optically pure 2-chloromandelic acid (ClMA) is a very important chiral drug intermediate for synthesis of (S)-clopidogrel, belonging to the platelet aggregation inhibitor. Enantioselective resolution of (R,S)-2-chloromandelic acid was carried out in organic solvent through irreversible transesterification catalyzed by lipase AK with vinyl acetate acting as the acyl donor. Effects of various conditions on enantioselectivity and activity of lipase were investigated, including organic solvents, temperature, water content, substrate ratio, enzyme loading, and reaction time. Based on homogeneous reaction and Ping-Pong bi-bi mechanism, a quantitative model was constructed to simulate and optimize the reaction process. Under the optimal conditions, excellent results were obtained with high conversion of (R)-2-ClMA (c _R, ≥98.85%) and large enantiomeric excess of substrate (ee _s, ≥98.15%). There is a good agreement between predicted values and experiment data, which indicates that the established method is a powerful tool for optimization of the enantioselective transesterification process for enantiomers separation.     

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.     

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.     

Immobilization of Candida rugosa lipase by cross-linking with glutaraldehyde followed by entrapment in alginate beads

Candida rugosa lipase was immobilized by first cross-linking with glutaraldehyde and then entrapping in calcium alginate beads. The presence of 2-propanol during cross-linking markedly improved the enzyme activity and activity recovery. Maximal enzyme activity (2.1?mmol?h^?1?g^?1 immobilized conjugate, wet weight) and activity recovery (117%) were observed at 30% (v/v) 2-propanol for hydrolysis of olive oil, which were 1.7 and 2.0 times higher than those of the immobilized enzyme prepared in the absence of 2-propanol. The half-life of the immobilized lipase prepared by entrapment after cross-linking in 30% 2-propanol was 1.6 times higher than that prepared by entrapment of the native lipase without cross-linking and 2-propanol pretreatment. The enantioselectivity of the former was 11 times higher than that of the latter for hydrolysis of racemic ketoprofen ethyl ester.