Enantioselective esterification of 2-methylbutyric acid catalyzed via lipase immobilized in microemulsion-based organogels

Chromobacterium viscosum lipase (c.v. lipase) was immobilized in microemulsion-based órganogels and successfully utilized for the enantioselective esterification of (+/?)-2-methylbutynic acid to preferentially form ethyl-(+)-2-methylbutyrate. The reaction time course and enantioselectivity obtained with the organogel—lipase system was compared and contrasted to that achieved in a reversed micellar solution system that contained lipase solubilized in its inner water core as well as that in which powdered lipases were directly dispersed in an organic solvent. The unique properties and potential benefits of the organogel system are discussed. © 1994 Wiley-Liss, Inc.     

Stereospecific hydrolysis of 3-(4-methoxyphenyl)glycidic ester in supercritical carbon dioxide by immobilized lipase

In the hydrolysis of racemic 3-(4-methoxyphenyl)glycidic acid methyl ester by immobilized Mucor miehel lipase in supercritical CO₂ the initial hydrolysis rate of the (2S,3R)-form was faster than the rate of the (2R,3S) -form. The stereoisomeric excess of the (2R,3S)-form reached 87 % at 53 % total conversion level. The water content of the reaction mixture and the initial concentration of the 3-(4-methoxyphenyl) glycidic acid methyl ester had no effect on isomeric purity. The reaction rate in supercritical CO₂ was considerably faster than in toluene/water -mixture.     

Continuous reaction-separation process for enzymatic esterification in supercritical carbon dioxide

The study of enzymatic esterification by an immobilized lipase in supercritical carbon dioxide (SCCO(2)) and in n-hexane, described in our previous works, was extended to continuous operation in a tubular fixed bed. The modeling of the reaction vessel operation was achieved through the use of the simple plug flow model coupled with the appropriate kinetic equation. Comparison with experiments proved to be satisfactory. The study of the postreactional separation, an important feature when using SCCO(2), was undertaken experimentally and good selectivities and product recovery were obtained. (c) 1994 John Wiley & Sons, Inc.     

Comparison of lipase-catalysed esterification in supercritical carbon dioxide and in n-hexane

Summary Oleic acid esterification by ethanol has been performed by an immobilized lipase fromMucor miehei in supercritical carbon dioxide and in n-hexane as solvents. In both media, determination of apparent kinetic constants has been achieved and influence of water content has been shown to be different due to various rates of water solubilities. Stability of the lipase has been proved to be correct and similar in both solvents. Inhibition by ethanol excess has been found but is greater in n-hexane. That can explain the higher initial velocities obtained in supercritical carbon dioxide for the highest ethanol concentrations.     

Development of magnetically separable immobilized lipase by using cellulose derivatives and their application in enantioselective esterification of ibuprofen

Highly active, stable, and magnetically separable immobilized enzymes were developed using carboxymethyl cellulose (CMC) and diethylaminoethyl cellulose DEAE-C; hereafter designated "DEAE" as supporting materials. Iron oxide nanoparticles penetrated the micropores of the supporting materials, rendering them magnetically separable. Lipase (LP) was immobilized on the surface of the supporting materials by using cross-linked enzyme aggregation (CLEA) by glutaraldehyde. The activity of enzyme aggregates coated on DEAE was approximately 2 times higher than that of enzyme aggregates coated on CMC. This is explained by the fact that enzyme aggregates with amine residues are more efficient than those with carboxyl residues. After a 96-h enantioselective ibuprofen esterification reaction, 6% ibuprofen propyl ester was produced from the racemic mixture of ibuprofen by using DEAE-LP, and 2.8% using CMC-LP.     

Enhanced biocatalytic activity of immobilized steapsin lipase in supercritical carbon dioxide for production of biodiesel using waste cooking oil

Bioprocess and Biosystems Engineering - The present work reports covalent immobilization of steapsin lipase (SL) on Immobead-350 support matrix (IMB) to make a robust biocatalytic system to work...     

Enantioselective esterification of ibuprofen by a novel thermophilic Biocatalyst: APE1547

The enantioselective esterification of ibuprofen catalyzed by a novel thermophilic esterase (APE1547) from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents. The effects of acyl acceptor, substrate ratio, organic solvent, temperature, and water activity were investigated. Under optimum conditions, the highest enantioselectivity (E = 38.1) was obtained with a higher enzyme activity (216.5 μmol/g/h). Celites were added into the reaction mixture to remove the water produced in the esterification. The reaction achieved its equilibrium in approximately 96 h with a conversion of 57 and 99% (ee) of the un-reacted (S)-ibuprofen obtained.     

Enantioselective synthesis of ibuprofen esters in AOT/isooctane microemulsions by Candida cylindracea lipase

The enantioselective esterification of racemic ibuprofen, catalyzed by a Candida cylindracea lipase, was studied in a water-in-oil microemulsion (AOT/isooctane). By using n-propanol as the alcohol, an optimal W(0) ([H(2)O]/[AOT] ratio) of 12 was found for the synthesis of n-propyl-ibuprofenate at room temperature. The lipase showed high preference for the S(+)-enantiomer of ibuprofen, which was esterified to the corresponding S(+)-ibuprofen ester. The R(-)-ibuprofen remained unesterified in the microemulsion. The calculated enantioselectivity value (E) for S-ibuprofen ester was greater than 150 (conversion 0.32). The enzyme activities of n-alcohols with different chain lengths (3-12) were compared, and it appeared that short- (propanol and butanol) and long-chained (decanol and dodecanol) alcohols were better substrates than the intermediate ones (pentanol, hexanol, and octanol). However, unlike secondary and tertiary alcohols, all of the tested primary alcohols were substrates for the lipase. The reversible reaction (i.e., the hydrolysis of racemic ibuprofen ester in the microemulsion) was also carried out enantioselectively by the enzyme. Only the S form of the ester was hydrolyzed to the corresponding S-ibuprofen. The reaction yield was, however, only about 4% after 10 days of reaction. The corresponding yield for the esterification of ibuprofen was about 35% (10 days). The high enantioselectivity displayed by the lipase in the microemulsion system was seen neither in a similar esterification reaction in a pure organic solvent system (isooctane) nor in the hydrolysis reaction in an aqueous system (buffer). The E value for S-ibuprofen ester in the isooctane system was 3.0 (conversion 0.41), and only 1.3 for S-ibuprofen in the hydrolysis reaction (conversion 0.32). The differences in enantioselectivity for the lipase in various systems are likely due to interfacial phenomena. In the microemulsion system, the water in which the enzyme is dissolved is separated from the solvent by a layer of surfactant molecules, thus creating an interface with a relatively large area. Such interfaces are not present in the pure organic solvent systems (no surfactant) nor in aqueous systems. (c) 1993 John Wiley & Sons, Inc.     

Subtilisin-catalyzed transesterification in supercritical carbon dioxide

Summary Subtilisin Carlsberg was found to catalyze transesterification between N-acetyl-L-phenylalanine chloroethyl ester and ethanol in supercritical carbon dioxide. The effects of different temperatures and carbon dioxide/ethanol ratios on the reaction rate were investigated. A comparative study showed that enzymatic transesterification is faster in supercritical carbon dioxide than in anhydrous organic solvents.     

Characteristics of immobilized lipase on hydrophobic superparamagnetic microspheres to catalyze esterification

A novel immobilized lipase (from Candida rugosa) on hydrophobic and superparamagnetic microspheres was prepared and used as a biocatalyst to catalyze esterification reactions in diverse solvents and reaction systems. The results showed that the immobilized lipase had over 2-fold higher activities in higher log P value solvents. An exponential increase of lipase activity against log P of two miscible solvent mixtures was observed for the first time. Both free and immobilized lipase achieved its maximum activity at the range of water activity (a(w)) 0.5-0.8 or higher. At a(w) 0.6, the immobilized lipase exhibited markedly higher activities in heptane and a solvent-free system than did the native lipase. In multicompetitive reactions, the alcohol specificity of the lipase showed a strong chain-length dependency, and the immobilized enzyme exhibited more preference for a longer-chain alcohol, which is different from previous reports. The immobilized lipase showed higher specificities for butyric acid and the medium-chain-length fatty acids (C(8)-C(12)). Then, the immobilized lipase was extended to solvent-free synthesis of glycerides from glycerol and fatty acids. Recovered by magnetic separation, the immobilized lipase exhibited good reusability in repeated batch reaction, indicating its promising feature for biotechnology application.     

二氧化硅纳米材料固定中性脂肪酶的条件优化及其特性

以二氧化硅纳米材料为载体,采用吸附法对脂肪酶进行固定化,研究了不同条件对固定化脂肪酶的催化活性的影响,得到最佳的固定化条件:给酶量为28300U/g,固定化温度为45oC,pH值为7.5,时间为10h,此时固定化酶的活力约为3867U/g载体。固定化酶的最适反应温度为45oC,比游离酶的反应温度高5oC,最适pH下降到5.5,低于游离酶的反应pH(pH7)。固定化酶的热稳定性和pH稳定性较游离酶有了很大的提高,其在70oC以下能保持70%以上的酶活力,而游离酶在50oC下残余酶活力仅为30%。在pH5～8的范围内,固定化酶的酶活力能保持50%以上,而游离酶只能保持20%左右。用固定化的中性脂肪酶催化不同的油品,即大豆油、菜籽油及泔水油生产生物柴油,菜籽油的酯化率最高。     

Extraction of squalene from yeast by supercritical carbon dioxide

Squalene produced under anaerobic conditions, by a strain of Torulaspora delbrueckii was extracted from the biomass using supercritical carbon dioxide. Minimum use of solvent, lower time of isolation and a higher selectivity of extraction merit use of supercritical fluid extraction (SFE) technique over solvent extraction of squalene, as optimized and reported previously. A maximum squalene yield of 11.12 g g^–1 (dry weight) of yeast cells was obtained at a temperature of 60 °C and pressure of 250–255 bar at a constant flow rate of 0.2l min^–1 of carbon dioxide. Lyophilization prior to SFE increased the squalene yield to 430.52 g g^–1 dry weight of yeast cells, an amount that is far greater than that obtained by (2:1) chloroform–methanol solvent extraction.     

Benzyl propionate synthesis by fed-batch esterification using commercial immobilized and lyophilized Cal B lipase

Bioprocess and Biosystems Engineering - In this work, a fed-batch approach was adopted to overcome propionic acid lipase inactivation effects in the benzyl propionate direct esterification mediated...     

超临界二氧化碳的四个应用方向

综述了超临界CO2的四个主要应用方向,即萃取、细胞破壁和微粒制造、反应溶剂以及SCCO2洗涤,着重总结了在烟草工业上的应用。对国内外的应用现状和应用前景给予了评述,并给出CO2温度、压力、密度的关系式。     

Effect of additives on the esterification activity of immobilized Candida antarctica lipase

In this work, the stabilizing effect of bovine serum albumin (BSA), peptone (PEP), and polyethylene glycol (PEG) during immobilization of Candida antarctica lipase on activated carbon was investigated. The influence of enzyme concentration and type of additive, added during the immobilization procedure, was studied using a 2² factorial central composite design. The goal was to maximize the synthetic activity of butyl butyrate, using butyric acid and butanol as substrate in n-heptane. An increase of 31–58% in the esterification activity was obtained when enzyme concentration on the supernatant was enhanced from 86.50 U m L⁻¹ to 226.80 U mL⁻¹. An enhancement in esterification activity of 38–68.95% was observed, depending on the initial enzyme concentration, when PEP was used instead of BSA. No significant increase in the esterification activity was observed when PEP was replaced by PEG. However, thermal stability tests at 50 °C showed that PEG had a higher stabilizing effect.     

The role of water on the equilibrium of esterification by immobilized lipase packed-bed column reactor

Summary The role of water on the continous synthesis of geranyl esters by immobilized lipase fromHumicola lanuginosa No. 3 was studied in a packed-bed column reactor (PBR) installed with a molecular sieve column for water extraction. The conversion degress by PBR were highly influenced by the water concentration throughout the reaction which acted as a determinant on the reaction equilibrium. Almost 100% conversion of geranyl laurate could be achieved under the optimum water-controlled conditions. The stability of the PBR system was for 35 days with the half-life of 38 days.     

Comparison of esterification and transesterification of fructose by porcine pancreas lipase immobilized on different supports

The syntheses of fructose butyrate with five different immobilized porcine pancreas lipases were studied in acetonitrile with butyric acid and tributyrin as acyl donors. The hydrolytic activities of the enzymes were measured during the syntheses. The transesterification yield was the highest with the Celite-adsorbed enzyme: 57% (1.42 g fructose butyrate l–1), and this form exhibited the highest degree of hydrolytic activity preservation (57%) after operation for 20 days.     

Biocatalytic esterification of citronellol with lauric acid by immobilized lipase on aminopropyl-grafted mesoporous SBA-15

Mesoporous SBA-15 was synthesized under acidic condition at 40 °C with a non-ionic triblock copolymer (P123) as the template. The synthesis gel composition used was 1 SiO₂:0.017 P123:2.9 HCl:202.6 H₂O. Functionalization of SBA-15 with 3-aminopropyltriethoxysilane (APTES) by post-synthesis method was performed under reflux for 2 h. The mesoporous samples were characterized using Fourier transform infrared (FT-IR), nitrogen adsorption, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). They were then utilized as supports for the immobilization of lipase to be subsequently used for the esterification of citronellol and lauric acid. Leaching and reusability tests were also conducted on the immobilized enzymes. Functionalization resulted in about 10% improvement in enzyme loading, leading to higher activity. The immobilized enzyme was also more stable to low pH and high temperature while showing better retention (up to 95%) of enzyme molecules. Immobilized lipase maintained 90% of its esterification activity in non-aqueous system even after 4 cycles of use. The improvements were associated with enhanced surface hydrophobicity, changes in pore shapes and stronger enzyme–support interactions with minimal effects to the enzymatic activity.     

Unfolding of poly-L-glutamic acid by microbubbling of supercritical carbon dioxide

The conformational changes in alpha-helical poly-L-glutamic acid caused by microbubbling supercritical CO2 were investigated with circular dichroism spectra. After microbubbling using a micropore filter at 35 and 30 MPa for 30 min, alpha-helix content decreased to 37%, while without the filter it was 68%. The alpha-helix structure was significantly decomposed by a high density of CO2. No important changes were observed in heating, autoclaving, or pH-lowering.     

Enzymatic dipeptide synthesis by surfactant-coated alpha-chymotrypsin complexes in supercritical carbon dioxide

Enzymatic dipeptide synthesis by surfactant-coated alpha-chymotrypsin complexes was performed in supercritical CO(2) and liquid CO(2) at 308.2 and 333.2 K at pressures of 6.1 and 10.1 MPa. The enzymatic activity of coated alpha-chymotrypsin complexes for dipeptides synthesis at 10.1 MPa in supercritical CO(2) (SC-CO(2)) was higher than that in a liquid CO(2) and ethyl acetate solution at 6.1 MPa. The behavior of alpha-chymotrypsin in SC-CO(2) was similar to that in liquid ethyl acetate. And increasing the pressure and temperature increased the maximum conversion and the enzymatic reaction rate in SC-CO(2). Furthermore, the control of the water content in the reaction media had a dominant effect on the enzymatic activity. The maximum conversion for the dipeptide synthesis by the surfactant-coated alpha-chymotrypsin was obtained at 4% water content. The alpha-chymotrypsin complexes exhibited a higher enzymatic activity than native alpha-chymotrypsin in SC-CO(2). The nonionic surfactants l-glutamic acid dialkyl ester ribitol amide and sorbitan monostearate were more favored than the anionic surfactant sodium bis(2-ethylhexyl)sulfosuccinate.