Influence of precursors and additives on microbial lipases stabilized by sol-gel entrapment

Sol-gel entrapment of microbial lipases from Candida cylindracea (Cc lipase),Pseudomonas fluorescens (Lipase AK), and Pseudomonas cepacia (Lipase PS), using as precursors tetraethoxysilane (TEOS) and silanes of type R-Si(OEt)₃ with alkyl or aryl R groups, has been investigated. Three different methods using these precursors were tried exhibiting protein immobilization yields in the range of 20–50%. Hydrolysis of emulsified olive oil, esterification of lauric acid with 1-octanol and enantioselective acylation of 2-pentanol have been used as model reactions for testing the properties of the encapsulated lipases. The recovery yields of the enzyme activity in the esterification reaction were between 20–68%, the best performance being achieved with phenyltriethoxysilane and tetraethoxysilane precursors at 3:1 molar ratio. When testing the entrapped Lipase AK in the enantioselective acylation reaction of 2-pentanol, activity recovery yields up to 32% related to the free enzyme were obtained and the immobilization increased the enantioselectivity of the enzyme.     

Immobilization of lipases onto the SBA-15 mesoporous silica

Lipase is an important enzyme which can catalyse the hydrolysis of lipids and has several applications and industrial potentials. In addition, different types of lipases are used as efficient catalysts in the enantioselective esterification and/or hydrolysis reactions and produce products in high yields and enantio excess as well. However, immobilization of lipases on the surface of a heterogeneous substrate is necessary to improve its specific catalytic activities as it can be isolated from the reaction mixture easily. Mesoporous silica materials are the best option for this aim due to their high specific surface area, ordered structure, and large pore volume. Hence, in this article, the role of SBA-15 and the modified SBA-15 mesoporous materials as support for different lipases and their catalytic activities are reviewed.     

Sol–gels and cross-linked aggregates of lipase PS from Burkholderia cepacia and their application in dry organic solvents

Lipase PS from Burkholderia cepacia (formerly Pseudomonas cepacia) was successfully immobilized in sol–gels under low methanol conditions using lyophilization in order to dry the gel. The enzyme was also cross-linked with glutaraldehyde to CLEAs without any additives. These immobilized enzyme preparations were employed for the highly enantioselective acylations of 1-phenylethanol (1), 1-(2-furyl)ethanol (2) and N-acylated 1-amino-2-phenylethanol (3) with vinyl acetate in organic solvents. Enzymatic hydrolysis of the obtained ester product was observed as a side reaction of the acylation of 3 in the presence of lipase PS powder. Hydrolysis was suppressed when the immobilized preparations of lipase PS were used.     

Immobilization of lipases for non-aqueous synthesis

Immobilization of lipases involves many levels of complications relating to the structure of the active site and its interactions with the immobilization support. Interaction of the so called hydrophobic ‘lid’ with the support has been reported to affect synthetic activity of an immobilized lipase. In this work we evaluate and compare the synthetic activity of lipases from different sources immobilized on different kinds of supports with varying hydrophobicity. Humicola lanuginosa lipase, Candida antarctica lipase B and Rhizomucor miehei lipase were physically adsorbed onto two types of hydrophobic carriers, namely hydrophilic carriers with conjugated hydrophobic ligands, and supports with base matrix hydrophobicity. The prepared immobilized enzymes were used for acylation of n-butanol with oleic acid as acyl donor in iso-octane with variable water content (0–2.8%, v/v) as reaction medium. Enzyme activity and effect of water on the activity of the immobilized derivatives were compared with those of respective soluble lipases and a commercial immobilized lipase Novozyme 435. Both R. miehei and H. lanuginosa immobilized lipases showed maximum activity at 1.39% (v/v) added water concentration. Sepabeads, a methacrylate based hydrophilic support with conjugated octadecyl chain showed highest immobilized esterification (synthetic) activity for all three enzymes, and of the three R. miehei lipase displayed maximum esterification activity comparable to the commercial enzyme.     

Pseudomonas cepacia lipase-catalysed resolution of racemic alcohols in ionic liquid using succinic anhydride: role of triethylamine in enhancement of catalytic activity

Racemic secondary alcohols were resolved via enantioselective acylation using succinic anhydride as acyl donor catalysed by lipase from Pseudomonas cepacia supported on celite (PS-C) in ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate [bmim]PF₆. Organic base, namely triethylamine as an additive in ionic liquid has been found to enhance the rate of the reaction.     

Influence of the alkyl-substituted silane precursor on sol–gel encapsulated lipase activity

Lipase from Candida rugosa was encapsulated within a chemically inert sol–gel support prepared by polycondensation of three precursor types (tetraethoxysilane (TEOS), methyltrimethoxysilane (MTMS) and polydimethylsilane (PDMS)) in the presence and absence of polyethylene glycol (PEG) and polyvinyl alcohol (PVA) as additives. Silica and their derivatives were characterised with regard to mean pore diameter, specific surface area, pore size distribution (BET method), weight loss upon heating thermogramivemetric analysis (TGA), chemical composition Fourier transform infrared spectroscopy (FT-IR), and catalytic activities. Immobilisation yields based on the recovered lipase activity vary from 3.02 to 31.98% and the highest efficiency was attained when lipase was encapsulated using TEOS in the presence of the PEG. Further information was obtained by testing the derivatives in esterification reactions and a different reactivity profile was found. Better performance was obtained with derivatives containing lipase encapsulated within gels prepared with MTMS as precursor in the presence of PEG. This lipase preparation exhibits increased esterification activity (155 μmol g⁻¹ min⁻¹), up to of three times greater than that prepared with TEOS (52 μmol g⁻¹ min⁻¹), and almost twice that prepared with MTMS/PDMS (89 μmol g⁻¹ min⁻¹) as precursors.     

Enzymatic fractionation of conjugated linoleic acid isomers by selective esterification

Attempt was done to prepare food supplements with high content of c9, t11-CLA or t10, c12-CLA. A free acid mixture containing CLA isomers was esterified with ethanol by enzyme catalysis. Novozyme 435 and Lipase AY30 were screened, and Lipase AY30 was employed to catalyze esterification reaction because of its high fractionation efficiency. Effect of reaction conditions on total esterification was investigated, and the optimal reaction conditions were: 140 U of lipase amount, reaction temperature at 50 °C, a pH of 6.5, and molar ratio of FFA–CLA to ethanol at 1:1. Based on the studies above, experiments of esterification and purification were done, and the best fractionation efficiency was obtained when the total esterification was 37%, and the corresponding purity and recovery of c9, t11-CLA were 75.50 and 49.85%, and that of t10, c12-CLA were 72.02 and 62.32%.     

Initial water content and lipase-mediated ester formation in hexane

Lipase biocatalysis was investigated as a tool for the production of esters by two model reactions, esterification of 1-butanol with 2-methyl-1-pentanoic acid and irreversible transesterification between 2-methyl-1-pentanol and vinyl acetate. The reactions were carried out in hexane using lipases from Candida cylindracea and porcine pancreas. The initial water content influenced both the yield of the ester and the enantioselectivity of the reaction (esterifica-tions) or the ester formation only (transesterifications).     

Disubstituted dialkoxysilane precursors in binary and ternary sol–gel systems for lipase immobilization

The disubstituted dimethyldiethoxysilane (DMDEOS), methyl(phenyl)diethoxysilane (MPDEOS) and diphenyldiethoxysilane (DPDEOS) were used in binary silane precursor systems in combination with tetraethoxysilane (TEOS) for the immobilization of lipase from Pseudomonas fluorescens (Lipase AK). In addition, ternary silane precursor systems with TEOS and octyltriethoxysilane (OTEOS) or phenyltriethoxysilane (PTEOS) were also studied for encapsulation. The best performing ternary sol–gel preparations (418–736% activity yields of the immobilized enzyme with 1-phenylethanol rac-1a as compared to the native form) were tested as biocatalysts for kinetic resolutions of rac-1a, 1-phenylpropan-2-ol rac-1b and 4-phenylbutan-2-ol rac-1c. Because the catalytic properties and the operational stability of the DMDEOS-containing preparations proved to be superior to all the tested free and sol–gel entrapped Lipase AK biocatalysts in batch mode, the kinetic resolutions of rac-1a and rac-1b were performed with the TEOS/PTEOS/DMDEOS 4:1:1 Lipase AK in a continuous-flow reactor as well.     

Screening and catalytic activity in organic synthesis of novel fungal and yeast lipases

A total of 969 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Ten lipase-producing microorganisms were selected and their taxonomic identification was carried out. From these strains Achremonium murorum, Monascus mucoroides, Arthroderma ciferri, Fusarium poae, Ovadendron sulphureo-ochraceum and Rhodotorula araucariae are described as lipase-producers for the first time. Hydrolysis activity of the crude lipases against both tributyrin and olive oil was measured. Heptyl oleate synthesis was carried out to test the activity of the selected lipases as biocatalysts in organic medium. All the selected lipases were tested as biocatalysts in several organic reactions using unnatural substrates. Lipases from the fungi Fusarium. oxysporum and O. sulphureo-ochraceum gave the best yields and enantioselectivities in the esterification of carboxylic acids. F. oxysporum and Penicillium chrysogenum lipases were the most active ones for the acylation of alcohols without steric hindrance. A. murorum lipase is very useful for the esterification of menthol. F. oxysporum and Fusarium. solani lipases were very stereoselective in the synthesis of carbamates.     

Immobilization of lipase on a new inorganic ceramics support, toyonite, and the reactivity and enantioselectivity of the immobilized lipase

A porous ceramics support, Toyonite 200-M (TN-M), for the immobilization of lipases was prepared hydrothermally from the minerals of kaolinite. Compared with some other commercial solid supports, the TN-M one exhibited better stability and higher selectivity for lipase proteins, and lipase PS (Pseudomonas cepacia) immobilized on the ceramics support showed higher reactivity for organic substrates than the free crude enzyme.     

Kinetic and modelling studies on the lipase catalysed enantioselective esterification of (±)-perillyl alcohol

Several lipases were kinetically studied with the aim to exploit their enantioselectivity in the esterification of (S)-(−) and (R)-(+)-perillyl alcohol with decanoic acid. Most of the lipases studied exhibited stereopreference towards the R-enantiomer with apparent E-values from 3.8 to 0.6, calculated as the initial esterification rates ratio for the individual enantiomers. In an attempt to interpret the structural basis of enantioselectivity, modelling studies were performed with two of these lipases, Candida cylindracea lipase (CcL) and Pseudomonas cepacia lipase (PcL) based on their previously determined X-ray crystal structures. The results derived from modelling studies confirm their stereopreferences towards the R-enantiomer, since increased conformational energy of the S-ester was found compared to the R-ester.     

Chemo-enzymatic preparation of resveratrol derivatives

Regioselective derivatisation of resveratrol (1) at positions 3, 5 or 4′ was achieved by a chemo-enzymatic procedure based on standard chemical reactions and esterification or alcoholysis in organic solvents catalysed by the commercially available Pseudomonas cepacia (PcL) and Candida antarctica (CaL) lipases.     

Lipase-catalyzed enantioselective acylation of 3-benzyloxypropane-1,2-diol in supercritical carbon dioxide

Lipase-catalyzed acylation of 3-benzyloxypropane-1,2-diol with vinyl acetate as acyl donor using different lipases [porcine pancreas lipase (PPL), Lipase AK “Amano”, Lipase PS “Amano”, and crude enzymes from Trichoderma reesei RUT-C30, Thermoascus thermophilus (NRRL5208), Talaromyces emersonii (NRLL3221)] was studied in supercritical carbon dioxide (scCO₂). In the reactions catalyzed by different lipases different amounts of monoacetate and diacetate products along with minor amounts of cyclic acetals forming from the diol and acetaldehyde were obtained.Application of Lipase AK led to the highest conversion (84.7%) and the highest enantiomeric excess values (ee_monoacetates = 38%, ee_diacetate = 85%). Effect of water content of scCO₂ on the productivity and the enantiomer selectivity of the reactions with Lipase AK was also investigated.     

Improvement of catalytic activity of lipase from Candida rugosa via sol-gel encapsulation in the presence of calix(aza)crown

Lipase from Candida rugosa (CRL) was encapsulated within a chemically inert sol-gel support in the presence of calix(aza)crowns as the new additives. The catalytic activity of the encapsulated lipases was evaluated both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic Naproxen methyl ester. It has been observed that the percent activity yields of the calix(aza)crown based encapsulated lipases were higher than that of the free lipase. Improved enantioselectivity was observed with the calix(aza)crown-based encapsulated lipases as compared to encapsulated free lipase. The reaction of Naproxen methyl ester resulted in 48.4% conversion for 24 h and 98% enantiomeric excess for the S-acid, corresponding to an E value of >300 (E = 166 for the encapsulated free enzyme). Moreover, the encapsulated lipases were still retained about 18% of their conversion ratios after the sixth reuse in the enantioselective reaction.     

Lipase-Catalyzed Synthesis of Capsaicin Analogs by Amidation of Vanillylamine with Fatty Acid Derivatives

Various lipases catalyzed the synthesis of capsaicin analogs by amidation of vanillylamine with fatty acid derivatives in a two-phase system. When methyl myristate was employed as an acyl donor, moderate yields, 40-59%, of capsaicin analog (3) were obtained using Novozym 435, Lipase AK or Lipase PS. Several capsaicin analogs having 4-18 carboxyl carbons were also synthesized from the corresponding fatty acid or its methyl or ethyl ester in 2-44% yields.     

Biocatalyst engineering exerts a dramatic effect on selectivity of hydrolysis catalyzed by immobilized lipases in aqueous medium

It has been found that enantioselectivity of lipases is strongly modified when their immobilization is performed by involving different areas of the enzyme surface, by promoting a different degree of multipoint covalent immobilization or by creating different environments surrounding different enzyme areas. Moreover, selectivity of some immobilized enzyme molecules was much more modulated by the experimental conditions than other derivatives. Thus, some immobilized derivatives of Candida rugosa (CRL) and C. antarctica-B (CABL) lipases are hardly enantioselective in the hydrolysis of chiral esters of (R,S)-mandelic acid under standard conditions (pH 7.0 and 25°C) (E<2). However, other derivatives of the same enzymes exhibited a very good enantioselectivity under nonstandard conditions. For example, CRL adsorbed on PEI-coated supports showed a very high enantio-preference towards S-isomer (E=200) at pH 5. On the other hand, CABL adsorbed on octyl-agarose showed an interesting enantio-preference towards the R-isomer (E=25) at pH 5 and 4°C. These biotransformations are catalyzed by isolated lipase molecules acting on fully soluble substrates and in the absence of interfacial activation against external hydrophobic interfaces. Under these conditions, lipase catalysis may be associated to important conformational changes that can be strongly modulated via biocatalyst and biotransformation engineering. In this way, selective biotransformations catalyzed by immobilized lipases in macro-aqueous systems can be easily modulated by designing different immobilized derivatives and reaction conditions.     

Covalent immobilization of pure isoenzymes from lipase of Candida rugosa

Covalent immobilization of pure lipases A and B from Candida rugosa on agarose and silica is described. The immobilization increases the half-life of the biocatalysts ( ) with respect to the native pure lipases ( ). The percentage immobilization of lipases A and B is similar in both supports (33–40%). The remaining activity of the biocatalysts immobilized on agarose (70–75%) is greater than that of the enzymatic derivatives immobilized on SiO₂ (40–50%). The surface area and the hydrophobic/hydrophilic properties of the support control the lipase activity of these derivatives. The thermal stability of the immobilized lipase A derivatives is greater than that of lipase B derivatives. The nature of the support influences the thermal deactivation profile of the immobilized derivatives. The immobilization in agarose (hydrophilic support) gives biocatalysts that show a greater initial specific reaction rate than the biocatalysts immobilized in SiO₂ (hydrophobic support) using the hydrolysis of the esters of (R) or (S) 2-chloropropanoic and of (R,S) 2-phenylpropanoic acids as the reaction test. The enzymatic derivatives are active for at least 196 h under hydrolysis conditions. The stereospecificity of the native and the immobilized enzymes is the same.     

Chemo-enzymatic synthesis of (R)-(+)-aminoglutethimide by kinetic resolution of (±)-4-cyano-4-phenyl-1-hexanol

Chemo-enzymatic approaches for the synthesis of the family of aromatase inhibitory drug via lipase-catalyzed kinetic resolution of (±)-4-cyano-4-phenyl-1-hexanol (2) as appropriate precursors were described. Enzymatic transesterification of primary alcohol (±)-2 using Pseudomonas cepacia (Amano PS, PCL) provided the enantiopure alcohol (R)-(−)-2 with 99% ee at conversion of 86%, while that of (±)-2 using Pseudomonas fluorescens (Amano AK, LAK) provided the (S)-(+)-2 with 96% ee at conversion of 86%. Chemical transformation of substrate (R)-(−)-2 gave (R)-(+)-aminoglutethimide (1) in enantioselectively high yield.     

Lipase-catalyzed optical resolution of trifluoro(aryl)ethanols

Optical resolutions of racemic 2,2,2-trifluoro-1-(aryl)ethanols — (1-naphthyl), (2-naphthyl), (4-methylnaphthyl), (phenyl), (1-pyrenyl) — were achieved by lipase-catalyzed enantioselective acetylations with vinyl acetate as an acetyl donor in octane, and (S)-acetates and (R)-alcohols were obtained. Among the lipases tested, lipase from Pseudomonas aeruginosa (lipase LIP, Toyobo) showed good enantioselectivity for above ethanols. However, no acetylation occurred with sterically hindered alcohols — (9-phenanthryl), (9-anthryl), (2-methylnaphthyl), (2, 4, 6-trimethylphenyl) — by various lipases. The resolutions of the three alcohols were carried out by the enantioselective alcoholysis or hydrolysis of their chloroacetates by lipase LIP.