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 racemic ketoprofen in organic solvents by lipase from Burkholderia cepacia G63

A lipase from the Burkholderia cepacia strain G63 immobilized on resin was used for the resolution of ketoprofen. To study its catalytic properties in enantioselective esterication, different alcohols and solvents were tested to select the most suitable acyl acceptor and reaction medium. Compared with the low activity of the free lipase, the enzyme activity and E value of the immobilized lipase were significantly enhanced. The enantioselectivity of the immobilized lipase could also be markedly improved by adding a small amount of 18-crown-6. RSM was employed to optimize the reaction parameters. The optimal reaction conditions were: reaction time 22.50 h, additives dosage 0.4322 g (0.33 mmol/mL), and substrate molar ratio 54.11:1. Under optimal conditions, the maximal E value was up to 10.01, which exhibited a better enantioselectivity than some commercial lipases, such as Novozym 435, Lipozyme RM IM and LipozymeTL IM.     

Efficient kinetic resolution of (R,S)-1-trimethylsilylethanol via lipase-mediated enantioselective acylation in ionic liquids

Efficient enantioselective acylation of (R,S)-1-trimethylsilylethanol {(R,S)-1-TMSE} with vinyl acetate catalyzed by immobilized lipase from Candida antarctica B (i.e., Novozym 435) was successfully conducted in ionic liquids (ILs). A remarkable enhancement in the initial rate and the enantioselectivity of the acylation was observed by using ILs as the reaction media when compared to the organic solvents tested. Also, the activity, enantioselectivity, and thermostability of Novozym 435 increased with increasing hydrophobicity of ILs. Of the six ILs examined, the IL C4MIm.PF6 gave the fastest initial rate and the highest enantioselectivity, and was consequently chosen as the favorable medium for the reaction. The optimal molar ratio of vinyl acetate to (R,S)-1-TMSE, water activity, and reaction temperature range were 4:1, 0.75, and 40 -50 degrees C, respectively, under which the initial rate and the enantioselectivity (E value) were 27.6 mM/h and 149, respectively. After a reaction time of 6 h, the ee of the remaining (S)-1-TMSE reached 97.1% at the substrate conversion of 50.7%. Additionally, Novozym 435 was effectively recycled and reused in C4MIm.PF6 for five consecutive runs without substantial lose in activity and enantioselectivity. The preparative scale kinetic resolution of (R,S)-1-TMSE in C4MIm.PF6 is shown to be very promising and useful for the industrial production of enantiopure (S)-1-TMSE.     

Determination of absolute configuration of secondary alcohols using lipase-catalyzed kinetic resolutions

Lipases show high enantioselectivity toward a wide range of secondary alcohols. An empirical rule based on the relative sizes of the substituents predicts which enantiomer reacts faster. X-ray structures of lipases provide a molecular basis for this empirical rule: their alcohol-binding pocket contains large hydrophobic pocket open to solvent and another smaller pocket. This predictable enantiopreference of lipases allows the determination of the absolute configuration of secondary alcohols using lipase-catalyzed kinetic resolution. Researchers have used this relative method to determine the configuration of approximately 50 secondary alcohols either as the only method or in combination with other methods.     

Optimization of (R,S)-1-phenylethanol kinetic resolution over Candida antarctica lipase B in ionic liquids

The kinetic resolution of racemates constitutes one major route to manufacture optically pure compounds. The enzymatic kinetic resolution of (R,S)-1-phenylethanol over Candida antarctica lipase B (CALB) by using vinyl acetate as the acyl donor in the acylation reaction was chosen as model reaction. A systematic screening and optimization of the reaction parameters, such as enzyme, ionic liquid and substrates concentrations with respect to the final product concentration, were performed. The enantioselectivity of immobilized CALB commercial preparation, Novozym 435, was assayed in several ionic liquids as reaction media. In particular, three different ionic liquids: (i) 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][PF₆], (ii) 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][BF₄] and (iii) 1-ethyl-3-methylimidazolium triflimide [emim][NTf₂] were tested. At 6.6% (w/w) of Novozym 435, dispersed in 9.520 M of [bmim][PF₆] at 313.15 K, using an equimolar ratio of vinyl acetate/(R,S)-1-phenylethanol after 3 h of bioconversion, the highest possible conversion (50%) was reached with enantiomeric excess for substrate higher than 99%.     

Lipase-catalysed kinetic resolution of secondary alcohols with improved enantioselectivity in propylene carbonate

The lipase-catalysed kinetic resolution of secondary alcohols was studied using vinyl acetate as acyl donor in propylene carbonate. Propylene carbonate offers an environmentally friendly alternative in contrast to conventional solvents. Several different lipases were investigated, and Candida antarctica lipase B (CALB) exhibited better results for all the substrates. It was shown that the addition of non-reactive base triethylamine and silver oxide to the reaction mixture enhanced the reaction rate and enantioselectivity. With propylene carbonate as solvent, CALB could be recycled without significant activity or enantioselectivity losses.     

有机相脂肪酶催化酮基各芬的立体选择性酯化

从５种脂肪酶中筛选出了具有较高催化活性和对映体选择性的脂肪酶Ｎｏｖｏｚｙｍ ４３５。进一步探讨了酶浓度、底物结构、底物浓度等因素对脂肪酶拆分酮基布洛芬（Ｋｅｔｏｐｒｏｆｅｎ）的影响。结果表明,以１０ｍＬ除水环己烷为反应介质,酶浓度为５ｍｇ／ｍＬ,Ｎｏｖｏｚｙｍ ４３５催化６．７ｍｍｏｌ／Ｌ Ｋｅｔｏｐｒｏｆｅｎ与２６．８ｍｍｏｌ／Ｌ丙醇进行酯化反应,反应３０ｈ,转化率为６８％时,Ｓ－酮基布洛芬对映     

超声波辅助下脂肪酶催化高酸值废油脂制备生物柴油

探讨了超声波辅助条件下脂肪酶催化高酸值废油脂转化为生物柴油的反应。来源于Aspergillus oryzae和Candida antarctica的固定化脂肪酶,在超声波辅助下,对高酸值废油脂转化为生物柴油具有高的催化活性。以来自于C.antarctica的固定化脂肪酶Novozym435为催化剂,以酸价为157mg KOH/g的高酸值废油脂为原料在超声波辅助下与丙醇反应,在脂肪酶用量为油质量的8%、初始醇油摩尔比为3∶1、反应温度控制在40~45℃、超声波频率和功率分别采用28kHz和100W的条件下,反应50min转化率达到94.86%。在此条件下,不同碳原子数(C1~C5)的直链和支链醇均有较高的转化率,在短链醇的选择上具有宽广的适应性。超声波还减少了反应产物和反应体系中其他黏性杂质在固定化脂肪酶表面的吸附,回收的Novozym435相较单纯机械搅拌条件下回收的外观干净、分散良好无结块现象、易于洗涤和再次利用,具有良好的操作稳定性。     

复合脂肪酶催化生物柴油的初步研究

初步探讨了复合脂肪酶催化生物柴油的工艺。优化了复合酶配比条件和叔丁醇反应体系。在无溶剂体系中,Novozym435分别与Lipozyme TLIM和Lipozyme RMIM均以70／30质量比混合时,甲酯得率分别达到94．52％和96．25％,比Novozym435单独催化时的甲酯得率分别提高了9．52％和9．99％。在叔丁醇体系中,当Novozym435与Li-pozyme TLIM和Lipozyme RMIM分别以60／40和80／20的质量比混合时,其甲酯得率分别为85．06％和81．5％,比Novozym435单独催化的效率分别提高了9．89％和7．48％。优化叔丁醇体系中复合酶催化条件后,甲酯得率达92％。     

Synthesis of methyl (R)-3-(4-fluorophenyl)glutarate via enzymatic desymmetrization of a prochiral diester

An efficient procedure for enzymatic desymmetrization of the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG) in an aqueous–organic phase was successfully developed to prepare methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG). Novozym 435 was selected as a highly efficient biocatalyst through lipase screening. The effects of various parameters in terms of co-solvent and its concentration, buffer pH, ionic strength and reaction temperature, on the reaction were investigated. It was found that 0.2 M phosphate buffer (pH 8.0) containing 20% MTBE (v/v) was the optimum reaction medium, and the optimum reaction temperature was 30 °C. Under the optimized reaction conditions, (R)-3-MFG was obtained in 95.6% ee value and 92.6% yield after 64 h when the concentration of 3-DFG and Novozym 435 were 200 mmol/l and 20 g/l respectively. Furthermore, Novozym 435 showed an excellent operational stability, retaining above 95% of the initial activity and enantioselectivity after 10 cycles of reaction. The developed method has a potential to be used for efficient enzymatic production of (R)-3-MFG.     

Efficient kinetic resolution of secondary alcohols using an organic solvent-tolerant esterase in non-aqueous medium

Objective

To identify an esterase-mediated kinetic resolution of secondary alcohols in non-aqueous medium.

Results

An esterase, EST4, from a marine mud metagenomic library, showed high activity and enantioselectivity for the kinetic resolution of secondary alcohols in non-aqueous medium. Using 1-phenylethanol as the model alcohol, the effects of organic solvents, acyl donors, molar ratio, temperatures and biocatalyst loading on the kinetic resolution catalyzed by the EST4 whole-cell biocatalyst were investigated and optimized. The optimized methodology was effective on resolving 16 various racemic secondary alcohols in neat n-hexane, providing excellent enantiomeric excess (up to 99.9 % ee). Moreover, EST4 exhibited a strong tolerance for high substrate concentration (up to 1 M), and the optical purity of the desired secondary alcohols was kept above 99 % ee.

Conclusion

The esterase EST4 is a promising biocatalyst for the enantioselective synthesis of various alcohols and esters with interesting practical applications.

     

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.     

Development of a magnetic biocatalyst useful for the synthesis of ethyloleate

Candida antarctica Lipase B was successfully immobilized on magnetite (Fe₃O₄) nanoparticles functionalized with chitosan and glutaraldehyde. The obtained magnetic catalyst was characterized and its performance was evaluated in solvent-free synthesis of ethyl oleate at room temperature. The performance of this biocatalyst was compared with the commercial Novozym 435, as a tool to estimate the efficiency of immobilization. It was found that using 33 mg of the biocatalyst it was possible to reach almost the same activity that was obtained using 12 mg of Novozym 435. Furthermore, this new biocatalyst presents the advantages of not being degraded by short alcohols, being easily recovered from the reaction media by magnetic decantation, and low fabrication cost. The possibility of reutilization was also studied, keeping a significant activity up to eight cycles. A special sampling protocol was also developed for the multiphasic reaction system, to assure accurate results. This novel biocatalyst is an interesting alternative for potential industrial applications, considering the above-mentioned advantages.     

Chemoenzymatic dynamic kinetic resolution of rac-1-phenylethanol in ionic liquids and ionic liquids/supercritical carbon dioxide systems

Continuous dynamic kinetic resolution processes in different ionic liquid/supercritical carbon dioxide biphasic systems were carried out by simultaneously using both immobilized Candida antarctica lipase B (Novozym 435) and silica modified with benzenosulfonic acid (SCX) catalysts at 40°C and 10 MPa. SCX was seen to act as an efficient heterogeneous chemical catalyst for the racemization of (S)-1-phenylethanol in different ionic liquid media ([emim][NTf₂], [btma][NTf₂] and [bmim][PF₆]). Coating both chemical and enzymatic catalysts with ILs greatly improved the efficiency of the process, providing a good yield (76%) of (R)-1-phenylethyl propionate product with excellent enantioselectivity (ee = 91–98%) in continuous operation.     

Production of human milk fat substitutes enriched in omega-3 polyunsaturated fatty acids using immobilized commercial lipases and Candida parapsilosis lipase/acyltransferase

In human milk fat (HMF), palmitic acid (20–30%), the major saturated fatty acid, is mostly esterified at the sn-2 position of triacylglycerols, while unsaturated fatty acids are at the sn-1,3 positions, conversely to that occurring in vegetable oils.This study aims at the production of HMF substitutes by enzyme-catalyzed interesterification of tripalmitin with (i) oleic acid (system I) or (ii) omega-3 polyunsaturated fatty acids (omega-3 PUFA) (system II) in solvent-free media. Interesterification activity and batch operational stability of commercial immobilized lipases from Rhizomucor miehei (Lipozyme RM IM), Thermomyces lanuginosa (Lipozyme TL IM) and Candida antarctica (Novozym 435) from Novozymes, DK, and Candida parapsilosis lipase/acyltransferase immobilized on Accurel MP 1000 were evaluated. After 24-h reaction at 60 °C, molar incorporation of oleic acid was about 27% for all the commercial lipases tested and 9% with C. parapsilosis enzyme. Concerning omega-3 PUFA, the highest incorporations were observed with Novozym 435 (21.6%) and Lipozyme RM IM (20%), in contrast with C. parapsilosis enzyme (8.5%) and Lipozyme TL IM (8.2%). In system I, Lipozyme RM IM maintained its activity for 10 repeated 23-h batches while for Lipozyme TL IM, Novozym 435 and C. parapsilosis enzyme, linear (half-life time, t_1/2 = 154 h), series-type (t_1/2 = 253 h) and first-order (t_1/2 = 34.5 h) deactivations were respectively observed. In system II, Lipozyme RM IM showed linear deactivation (t_1/2 = 276 h), while Novozym 435 (t_1/2 = 322 h) and C. parapsilosis enzyme (t_1/2 = 127 h), presented series-type deactivation. Both activity and stability of the biocatalysts depended on the acyl donor used.     

Lipase-catalyzed kinetic resolutions of racemic β- and γ-thiolactones

Several racemic β- and γ-thiolactones were synthesized and kinetic resolutions of them were executed using lipases. While a lipase from Pseudomonas cepacia (PCL) showed the highest enantioselectivity for (S)-form (>99% ee_S at 53% conversion, E > 100) in the kinetic resolution of racemic -methyl-β-propiothiolactone (rac-MPTL), it showed no hydrolysis activity in the kinetic resolution of -benzyl--methyl-β-propiothiolactone (rac-BMPTL), suggesting that the changes in the size of alkyl group from rac-MPTL to rac-BMPTL leads to lower hydrolysis activity and enantioselectivity. In contrast, racemic γ-butyrothiolactones were hydrolyzed by several lipases with low enantioselectivity, whereas a lipase from Candida antarctica (CAL) showed moderate enantioselectivity for (S)-form (>99% ee_S at 76% conversion, E = 11) in the kinetic resolution of racemic -methyl-γ-butyrothiolactone (rac-MBTL). Computer-aided molecular modeling was also performed to investigate the enantioselectivites and activities of PCL toward β-propiothiolactones. The computer modeling results suggest that the alkyl side chains of β-propiothiolactones and γ-butyrothiolactones interact with amino acid residues around hydrophobic crevice, which affects the activity of PCL.     

Combining regio- and enantioselectivity of lipases for the preparation of (R)-4-chloro-2-butanol

Preparation of 98% ee (R)-4-chloro-2-butanol was carried out by the enzymatic hydrolysis of chlorohydrin esters, using fungal resting cells and commercial enzymes. Hydrolyzes were carried out using lipases from Candida antarctica (Novozym 435), C. rugosa, Rhizomucor miehei (Lipozyme IM), Burkolia cepacia, and resting cells of Rhizopus oryzae and Aspergillus flavus. The influence of the enzyme, the solvent, the temperature, and the alkyl chain length on the selectivity of hydrolyzes of isomeric mixtures of chlorohydrin esters is described. Regioselectivity was higher than 95% for some of the tested lipases. Novozym 435 allowed preparation of the (R)-4-chloro-2-butanol after 15 min of reaction at 30-40 degrees C.     

Lipase catalysed synthesis of sebacic and phthalic esters

The enzymatic synthesis and hydrolysis of alkyl sebacates and o-, m-, p-phthalates were studied. Biosyntheses were conducted through alcoholysis of dimethyl phthalates and dimethyl sebacate with 2-ethylhexanol and 3,5,5-trimethylhexanol in a solvent-free medium, using lipases from Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme IM) and Porcine pancreas (PPL). It was found that the synthesis and hydrolysis of sebacic acid esters were characterised by a satisfactory rate, however, by low enantioselectivity. The yield of synthesis of di-3,5,5-trimethylhexyl sebacate catalysed by Novozym 435 at 50 °C was 84%, after 20 h of reaction. The degree of conversion, 62.9% after 350 h, was obtained for alcoholysis reaction of dimethyl m-phthalate with 3,5,5-trimethylhexanol. For the enzymes used, no activity was detected at all on both the synthesis and hydrolysis of di-2-ethylhexyl o-phthalate and di-3,5,5-trimethylhexyl o-phthalate.     

Immobilized lipase-catalysed synthesis of cinnamyl laurate in non-aqueous media

Esters of cinnamyl alcohol find many applications in food, cosmetic and pharmaceutical industries as flavor and fragrance compounds. The current work focuses on the synthesis of cinnamyl laurate from cinnamyl alcohol and lauric acid, including screening of various immobilized lipases and optimization of reaction conditions such as catalyst loading, speed of agitation, mole ratio and temperature. Among different lipases screened such as Novozym 435, Lipozyme RM IM and Lipozyme TL IM, Novozym 435 was found to be the best catalyst with 60% conversion in 2 h at 30 °C for equimolar quantities of the reactants using 0.33% (w/v) of catalyst and toluene as solvent. An ordered bi–bi mechanism with dead-end complex of lauric acid was found to represent the kinetic data.     

Novozym 435 displays very different selectivity compared to lipase from Candida antarctica B adsorbed on other hydrophobic supports

This paper shows that the properties of lipase B from Candida antarctica (CAL-B) may be easily modulated using different hydrophobic supports to immobilize it (octyl and butyl-agarose, octadecyl-Sepabeads or Lewatit). CAL-B could be fully desorbed from the supports by just incubating the biocatalyst with Triton X-100, although the concentration of detergent necessary was to fully desorb the enzyme varied with the support employed (from 1% for butyl-agarose to 4% for octadecyl-Sepabeads), suggesting that in all cases, the main reason for the enzyme immobilization was hydrophobic interactions. Lewatit VP OC 1600 yielded very different results in terms of activity, selectivity or enantioselectivity in the hydrolysis of rac-2-O-butyryl-2-phenylacetic acid (1) and 3-phenylglutaric acid dimethyl diester (3) compared to the other preparations. For example, in the hydrolysis of 1, Novozym 435 preferred the S-isomer (with an E value higher than 100) whereas all the other preparations preferred the R isomer (e.g. octyl-agarose-CAL-B with E value of 50). In the hydrolysis of 3, Novozym 435 gave S-3-phenylglutaric acid methyl ester with an ee higher than 99%, by coupling the first asymmetric hydrolysis to the enantiospecific hydrolysis of the monoester. CAL-B immobilized on Lewatit at low ionic strength not only behaved similarly to Novozym 435, but also presented some differences that should be due to the exact protocol of the enzyme immobilization in Novozym 435.