Esterification In Organic Solvents: Selection Of Hydrolases And Effects Of Reaction Conditions

Fifty different hydrolases were screened for retention of high esterification activity in an organic solvent with citronellol as substrate. Although 22 hydrolases were very active as catalysts in the organic solvent, lipase from Candida cylindracea (lipase OF 360) was selected for further examination of the effects of reaction conditions on enzyme activity, with regard to catalyst availability and activity retention after immobilization. When the enzyme was entrapped in hydrophobic polyurethane gels, water-saturated isooctane was found to be the most suitable solvent, whereas polar solvents caused reversible catalyst inactivation. Entrapment significantly enhanced the operational stability of the lipase in the organic solvent.     

Immobilization by Adsorption of Hydrophobic Lipase Derivatives to Porous Polymer Beads for Use in Ester Synthesis

A novel procedure for attaching lipase to certain kinds of hydrophobic surfaces is described. The procedure involves covalent derivatization of the protein molecule by reaction in solution with a hydrophobic imidoester, aldehyde or activated polyethylene glycol. The resulting protein derivative is then allowed to adsorb onto an insoluble hydrophobic surface. Quantitative adsorption is observed and the enzyme is bound very strongly on the support The number and nature of the hydrophobic substituents introduced in the chemical derivatization step can be easily controlled. The adsorption step occurs spontaneously upon exposure of the modified protein to a variety of hydrophobic materials. The hydrophobic lipase derivative obtained by reaction with PEG activated with p-nirrophenyl chloroformate, for example, adsorbs readily onto polyacrylate and polystyrene beads, with most of its esterification activity in organic solvent intact. Its thermostability is also greatly enhanced. Derivatization of lipase with hydrophobic groups greatly enhances its esterification activity in organic solvent, and its immobilization in this manner enables the preparation of a highly reactive biocatalyst for biotechnological application.     

Immobilization and activity of Rhizomucor miehei lipase. Effect of the matrix properties prepared from nonionic fluorinated surfactants

We report the immobilization of Rhizomucor miehei lipase (RmL) onto mesoporous silica materials, in particular the investigations concerning the effects of the level of silica condensation and of the pore size on the enzyme activity. The efficiency of the immobilization was revealed by FTIR spectroscopy. Infrared was also used to determine the quantity of adsorbed enzyme. Immobilization efficiency increased when the RmL concentration in the buffer solution was changed from 2 to 10 mg/mL. Nevertheless, while upon enzyme immobilization the mesopore ordering was sustained for the support recovered after hydrothermal treatment at 100 °C, a structure collapse occurred for the one prepared at 80 °C. The difference in behavior is attributed to the lower hydrothermal stability of this material, which reflects the lower level of silica condensation. The enzyme-containing mesostructured silica was effectively used to catalyze the model esterification reaction of lauric acid with 1-propanol, as the immobilized lipase retained its catalytic activity. A linear relationship was observed between the reaction rate and the amount of catalyst. RmL immobilized on mesoporous materials presented a satisfactory reusability, while the remaining activity of RmL after 4 months of storage was 47% of the initial one.     

Efficient immobilization of lipases by entrapment in hydrophobic sol-gel materials

The commercial application of lipases as biocatalysts for organic synthesis requires simple but efficient methods to immobilize the enzyme, yielding highly stable and active biocatalysts which are easy to recover. In this study, we present a novel method to achieve lipase immobilization by entrapment in chemically inert hydrophobic silica gels which are prepared by hydrolysis of alkyl-substituted silanes in the presence of the enzyme. A typical immobilization procedure uses: an aqueous solution of lipase; sodium fluoride as a catalyst; and additives like polyvinyl alcohol or proteins and alkoxysilane derivatives like RSi-(OMe)(3) with R = alkyl, aryl, or alkoxy as gel precursors. The effect of various immobilization parameters like stoichiometric ratio of water, silane, type and amount of additive, type and amount of catalyst, and type of silane has been carefully studied. The new method is applicable for a wide variety of lipases, yielding immobilized lipases with esterification activities enhanced by a factor of up to 88, compared to the commercial enzyme powders under identical conditions. Studies on the stability of sol-gel immobilized lipases under reaction conditions or storage (dry, in aqueous or organic medium) revealed an excellent retention of enzymatic activity. The possible reasons for the increased enzyme activities are discussed. (c) 1996 John Wiley & Sons, Inc.     

Electrospun polyacrylonitrile nanofibrous membranes for lipase immobilization

Polyacrylonitrile (PAN) nanofibers could be fabricated by electrospinning with fiber diameter in the range of 150–300 nm, providing huge surface area for enzyme immobilization and catalytic reactions. Lipase from Candida rugosa was covalently immobilized onto PAN nanofibers by amidination reaction. Aggregates of enzyme molecules were found on nanofiber surface from field emission scanning electron microscopy and covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After 5 min activation and 60 min reaction with enzyme-containing solution, the protein loading efficiency was quantitative and the activity retention of the immobilized lipase was 81% that of free enzyme. The mechanical strength of the NFM improved after lipase immobilization where tensile stress at break and Young's modulus were almost doubled. The immobilized lipase retained >95% of its initial activity when stored in buffer at 30 °C for 20 days, whereas free lipase lost 80% of its initial activity. The immobilized lipase still retained 70% of its specific activity after 10 repeated batches of reaction. This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters.     

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.     

Mass transfer effects in solvent-free fat interesterification reactions: influences on catalyst design

The use of solvent-free systems in the oil and fats industry is commonplace. Initial studies on interesterification were carried out in solvent systems because the lipase was immobilized solely by adsorption onto particles of diatomaceous earth. In this study, the mass transfer characteristics associated with the continuous interesterification of olive oil in a solvent-free system have been examined, for lipase immobilized on the three ion-exchange materials: Duolite ES562, Duolite ES568, and Spheroil DEA. The process of immobilization is influenced by the internal structure of the material and this in turn influences the interesterification activity of the catalyst. Individually prepared catalysts for the three support materials have shown that external mass transfer limitations are unlikely even at low flowrates.In the case of Spherosil DEA, with a mean pore diameter of 1480 A, the wide pores would be expected to reduce internal mass transfer limitations; however, it is more likely that the reduction in activity with increased catalyst loading is due to the lipase molecules being immobilized in a tightly packed monolayer. In such a situation, some active sites of the lipase molecules would become inaccessible to substrate molecules leading to an observed reduction in activity. For Duolite ES568, the observed results are very similar to those seen for Spherosil DEA, however, the pore structure of this support material indicate that some internal mass transfer limitations may also be occurring. Yet the contribution of the individual effects cannot be determined. The results observed for the support Duolite ES562 are different than those observed for the other materials and reflect the heterogeneity of Duolite ES562. The large proportion of narrow pores in the support mean that, for the catalysts examined, immobilization is most likely to have occurred in the external pores of the particles, and as such no internal mass transfer limitation is observed.It is clear that for interesterification the material chosen for enzyme immobilization will have an important role in determining the catalyst efficiency. External mass transfer limitations are very minor and observed internal mass transfer limitations may be caused by both internal mass transfer and the manner in which the immobilization process occurs. (c) 1994 John Wiley & Sons, Inc.     

离子液体中固定化脂肪酶催化拆分（±）-薄荷醇

以自制的平均粒径为4．5um磁性高分子微球为载体,采用离子交换法固定化Candida rugosa脂肪酶,催化（±）-薄荷醇的酯化反应,以考察反应时间、pH、反应温度、水活度等因素对酶的固定化以及酯化反应的影响。在固定化反应150min、pH5．0、酯化反应温度30℃、固定化酶的水活度为0．78的条件下,所制备的固定化脂肪酶在离子液体[bmim]PF6中催化拆分（±）-薄荷醇的效果最佳,与游离酶相比固定化脂肪酶的立体选择性有很大的提高,对映体过量率可达93％,对映体选择值为35。     

Immobilization of lipase on hydrophobic nano-sized magnetite particles

As a tool for the stable enzyme reuse, enzyme immobilization has been studied for several decades. Surface-modified nano-sized magnetite (S-NSM) particles have been suggested as a support for the immobilization of enzyme in this study. Based on the finding that a lipase is strongly adsorbed onto a hydrophobic surface, NSM particles (8–12 nm) were made hydrophobic by binding of sodium dodecyl sulfate via a sulfate ester bond. Various types of measurements, such as transmission electron microscopy, X-ray diffraction, infrared spectroscopy, vibration sample magnetometer, and thermo gravimetric analysis, were conducted in characterizing S-NSM nanoparticles. S-NSM particles were used for the adsorption of porcine pancreas lipase (PPL). A dodecyl carbon chain is expected to form a spacer between the surface of the NSM and the lipase adsorbed. The immobilized PPL showed the higher specific activity of oil hydrolysis than that of free one. Immobilized PPL could be recovered by magnetic separation, and showed the constant activity during the recycles.     

Biodiesel fuel production via transesterification of oils using lipase biocatalyst

Biodiesel has gained widespread importance in recent years as an alternative, renewable liquid transportation fuel. It is derived from natural triglycerides in the presence of an alcohol and an alkali catalyst via a transesterification reaction. To date, transesterification based on the use of chemical catalysts has been predominant for biodiesel production at the industrial scale due to its high conversion efficiency at reasonable cost. Recently, biocatalytic transesterification has received considerable attention due to its favorable conversion rate and relatively simple downstream processing demands for the recovery of by-products and purification of biodiesel. Biocatalysis of the transesterification reaction using commercially purified lipase represents a major cost constraint. However, more cost-effective techniques based on the immobilization of both extracellular and intracellular lipases on support materials facilitate the reusability of the catalyst. Other variables, including the presence of alcohol, glycerol and the activity of water can profoundly affect lipase activity and stability during the reaction. This review evaluates the current status for lipase biocatalyst-mediated production of biodiesel, and identifies the key parameters affecting lipase activity and stability. Pioneer studies on reactor-based lipase conversion of triglycerides are presented.     

Amino silicones finished fabrics for lipase immobilization: Fabrics finishing and catalytic performance of immobilized lipase

Finishing of silk fabric was achieved by using amino-functional polydimethylsiloxane (PDMS) and lipase from Candida sp. 99-125 was immobilized on the treated silk fabrics. Hydrophobic fabrics were obtained by dipping the native fabric in 0.125–0.25% (w/v) PDMS solution and dried at 70 °C. The direct adsorption on PDMS-treated fabric was verified to be a better strategy for lipase immobilization than that by covalent binding. Compared to unfinished fabrics, the hydrolytic activity of immobilized enzyme on the finished fabric was improved by 1.6 times. Moreover, the activity of immobilized enzymes on hydrophobic fabrics was significantly improved in different concentrations of strong polar solvents such as methanol and ethanol, and in common organic solvents with different octanol–water partition coefficients (Log P). Enzymatic activity and stability in 15% water content system (added water accounted for the total reaction mixtures, v/v) showed more than 30% improvement in each batch. The amino–silicone finished fabric surface was investigated by scanning electron microscopy and X-ray photoelectron spectroscopy. The hydrophobic fabric immobilized enzyme could be recycled for more than 80 times with no significant decrease in esterification activity. PDMS-treated woven silk fabrics could be a potential support for lipase immobilization in catalytic esterification processes.     

Immobilization of lipase into mesoporous silica particles by physical adsorption

Mesoporous silica particles for immobilization of lipase from Candida rugosa were prepared by precipitation and aggregation of primary particles from highly basic sodium silicate solution but without addition of templates. The average pore size of the material was 15.8 nm, which allowed enzyme adsorption inside the pores and high enzyme loading. Specific surface area of the material was found to be 359 m²g^?1. A loading of 100 mglipasegdrysilica^?1 was obtained at initial enzyme concentration of 1.8 mgmL^?1 by physical adsorption. The FTIR spectrum showed the structural conformation of lipase to be retained after adsorption into the mesoporous silica support. Although the efficiency of the mesoporous biocatalyst was shown to be lower than that of the free enzyme, the immobilized enzyme showed enhanced thermal stability and could be desorbed with Triton X-100, indicating the hydrophobic nature of the adsorption.     

Immobilization of lipase Eversa Transform 2.0 on poly(urea–urethane) nanoparticles obtained using a biopolyol from enzymatic glycerolysis

In this work, the free lipase Eversa^® Transform 2.0 was used as a catalyst for enzymatic glycerolysis reaction in a solvent-free system. The product was evaluated by nuclear magnetic resonance (¹H NMR) and showed high conversion related to hydroxyl groups. In sequence, the product of the glycerolysis was used as stabilizer and biopolyol for the synthesis of poly(urea–urethane) nanoparticles (PUU NPs) aqueous dispersion by the miniemulsion polymerization technique, without the use of a further surfactant in the system. Reactions resulted in stable dispersions of PUU NPs with an average diameter of 190 nm. After, the formation of the PUU NPs in the presence of concentrated lipase Eversa^® Transform 2.0 was studied, aiming the lipase immobilization on the NP surface, and a stable enzymatic derivative with diameters around 231 nm was obtained. The hydrolytic enzymatic activity was determined using ρ-nitrophenyl palmitate (ρ-NPP) and the immobilization was confirmed by morphological analysis using transmission electron microscopy and fluorescence microscopy.

     

Evaluation of a silica-coated magnetic nanoparticle for the immobilization of a His-tagged lipase

Magnetic particles of size 10 nm have been coated with silica to a mean diameter of 40 nm and charged with Cu²⁺ ions via a multidentate ligand, iminodiacetic acid (IDA), for the immobilization of His-tagged Bacillus stearothermopilus L1 lipase. Microporous (average pore diameter of 60 Å) silica gel with a mean particle diameter of 115 µm has been used as a comparative support material. The molar ratio of Cu²⁺ to IDA was found to be 1:1.14 and 1:1.99 in the silica gel and the silica-coated magnetic nanoparticles (SiMNs), respectively. The specific activity of the immobilized enzyme was found to conform to the following order: Cu²⁺-charged SiMN>SiMN>Cu²⁺-charged silica gel>silica gel. When it was immobilized on the Cu²⁺-charged SiMNs, over 70% of the initial activity of the lipase remained after it had been reused five times. However, only 20% of the initial activity remained after the enzyme immobilized on the Cu²⁺-charged silica gel had been reused five times. For the enzyme immobilized on supports without Cu²⁺ cations, all activity was lost after threefold reuse. The differences in the specific activities and the efficiencies of reuse of the enzymes immobilized on the various support materials are discussed in terms of immobilization mechanisms (physical adsorption vs. coordination bonding), mass transfer of a substrate and a product of the enzyme reaction, and the status of the Cu (Cu bound to the IDA on the silica layer vs. Cu directly adsorbed on the silica layer).     

Biocatalysis using gelatin microemulsion-based organogels containing immobilized Chromobacterium viscosum lipase

Chromobacterium viscosum (CV) lipase was immobilized in gelatin-containing Aerosol-OT (AOT) microemulsion-based organogels (MBGs). The behavior of this novel, predominantly hydrophobic matrix as an esterification catalyst has been examined. The biocatalyst was most effective when the MBG was granulated to yield gel particles of approximately 500 mum diameter, providing a total surface area of ca. 10(6) mm(2) per 10 cm(3) of gel. The gel was generally contacted with a solution of the substrate(s) in a hydrocarbon oil. Under most conditions reaction was not diffusion limited. Apparent lipase activity was influenced by certain compositional changes in the MBG, but most significantly when the R value, the mole ratio of water to surfactant, was altered. Higher activities were observed at lower R values. Although gels of lowest R value expressed the highest condensation activity, such formulations were physically unsuitable as immobilization matrices due to their proximity to the gel-solution phase boundary. MBGs of intermediate R values (between 60 and 80) were considered most suitable because they offer relatively high condensation activity and good physical stability. The gelatin concentration also exerted a small but measurable influence on the observed condensation rates. Apparent lipase activity was also influenced to some extent by the nature of the parent hydrocarbon used to prepare the MBG. Higher activities were obtained using formulations derived from isooctane and cyclohexane rather than the n-alkanes. Condensation activities expressed by CV lipase in the MBGs were broadly comparable to those expressed in the analogous parent water-in-oil (w/o) microemulsions. The MBGs functioned effectively in neat substrate solutions, but the condensation activity expressed by the MBGs in a series of successive batch syntheses was adversely affected by the formation and retention of the water coproduct. Selective removal of the water was achieved using a concentrated solution of dry reverse micelles, which resulted in recovery of lost activity. Pretreatment of lipase-containing MBGs resulted in the formation of MBGs with enhanced catalytic properties and modified composing the conventional procedure. (c) 1997 John Wiley & Sons, Inc.     

Immobilization of enzymes into nanocavities for the improvement of biosensor stability

Nanoporous materials with different pore sizes are evaluated as immobilization and stabilization matrices of proteins for the development of highly stable biosensors. It has been proven experimentally that confinement of proteins in cages with a diameter that is 2-6 times larger than their size increases considerably the stability of the biomolecules, as has been shown earlier by theoretical calculations. Porous silica beads with pore sizes of 10nm were utilized for the immobilization of the enzymes HRP and GOx with diameters in the order of 5 and 7 nm, respectively. The sensitivity of the corresponding biosensor systems was monitored for 70 h under continuous operation conditions (+600 mV) and it was found that the stabilization factor of GOx is 1.7 times higher compared to HRP. Also the stabilization efficiency of enzymes against leaching and inactivation in porous polymer beads with pore diameters of 10 and 30 nm was examined. The leaching rate of the enzyme AChE from the 30 nm polymer beads was found to be 1.1 times higher than that from the 10nm beads. At the same time the remaining activity of GOx biosensors after 5 days of continuous operation conditions (+600 mV) was found to be 2.1 times higher when the enzyme had been immobilized in the 10nm beads compared to the 30 nm beads. It is thus evident that the matching between the pore size of nanoporous materials and the molecular size of enzymes is essential for the development of biosensors with extended shelf and operational lifetimes.     

Effect of Different Supports on the Reaction Rate of Rhizomucor Miehei Lipase in Organic Media

Five different aluminas, a silica and a zirconia support were used to adsorb lipase (E.C. 3.1.1.3) from Rhizomucor miehei. The activity of the immobilised lipase was measured by esterification of dodecanol and decanoic acid in hexane. The immobilised lipase and the organic phase were pre-equilibrated separately to known water activities before mixing them to commence the reactions. The aluminas, which varied in pore sizes and surface areas, adsorbed similar amounts of enzyme. However, the esterification activities varied about 10-fold, increasing with increasing surface area. The silica and zirconia supports adsorbed about half as much lipase as the aluminas. The esterification reaction rates per unit quantity of enzyme adsorbed were compared with those for aluminas with similar surface areas; this specific rate was about 2 times higher for the zirconia, but the difference with silica was only small. There was no clear correlation between the esterification rates at fixed water activity and the amount of water adsorbed by the support used.     

Solid-phase handling of hydrophobins: immobilized hydrophobins as a new tool to study lipases

Hydrophobins are fungal proteins that self-assemble spontaneously at hydrophilic-hydrophobic interfaces and change the polar nature of the surfaces to which they attach. This attribute can be used to introduce hydrophobic foci on the surface of hydrophilic supports where hydrophobins are attached by covalent binding. In this paper, we report the binding of Pleurotus ostreatus hydrophobins to a hydrophilic matrix (agarose) to construct a support for noncovalent immobilization and activation of lipases from Candida antarctica, Humicola lanuginosa, and Pseudomonas flourescens. Lipase immobilization on agarose-bound hydrophobins proceeded at very low ionic strength and resulted in increased lipase activity and stability. The enzyme could be desorbed from the support using moderate concentrations of Triton X-100, and its enantioselectivity was similar to that of lipases interfacially immobilized on conventional hydrophobic supports. These results suggest that lipase adsorption on hydrophobins follows an "interfacial activation" mechanism; immobilization on hydrophobins offers new possibilities for lipase study and modulation and reveals a new application for fungal hydrophobins.     

Design and synthesis of lipase nanogel with interpenetrating polymer networks for enhanced catalysis: Molecular simulation and experimental validation

A temperature-responsive lipase nanogel (denoted as CRL-IPN nanogel), in which lipase is encapsulated into an interpenetrating polymer matrix formed by polyacrylamide and poly(N-isopropylacrylamide) (PNIPAAm) has been designed and synthesized for an enhanced stability and activity in both aqueous and non-polar organic solvents. A three-step method, including acryloylation, polymerization with acrylamide and sequential polymerization with N-isopropylacrylamide, was established to fabricate enzyme nanogel with temperature-sensitive interpenetrating polymer network. It has been shown by an all-atom molecular dynamics simulation that above mentioned polymer matrix forms a more hydrophobic environment, as compared to that obtained with sole polyacrylamide, because of the penetration of N-isopropylacrylamide into the polymer acrylamide network via hydrogen bonding, which is further confirmed by the fluorescence spectrum. This favours the uptake of hydrophobic substrates and thus the overall rate of enzymatic catalysis. The enhanced stability and catalytic performance of this novel lipase nanogel in aqueous and non-polar organic solvent were demonstrated by using hydrolysis reaction of p-NPP in aqueous and esterification reaction of ibuprofen in isooctane. In aqueous solution, the residual activity of CRL-IPN nanogel maintains its 70% activity at 60 °C after 4 h, compared with that free lipase only has 30% at the same condition. In addition, the CRL-IPN nanogel can be reused for 10 cycles with no loss of its activity. In isooctane, CRL-IPN nanogel gave a 33% yield of esterification of ibuprofen, in comparison to 22% using free lipase and less than 5% using lipase encapsulated in a polyacrylamide matrix. The enhanced stability and activity make this CRL-IPN nanogel promising for enzymatic catalysis in non-polar solvents.     

Alginate as immobilization matrix and stabilizing agent in a two-phase liquid system: application in lipase-catalysed reactions.

Alginate was evaluated as an immobilization matrix for enzyme-catalyzed reactions in organic solvents. In contrast to most hydrogels, calcium alginate was found to be stable in a range of organic solvents and to retain the enzyme inside the gel matrix. In hydrophobic solvents, the alginate gel (greater than 95% water) thus provided a stable, two-phase liquid system. The lipase from Candida cylindracea, after immobilization in alginate beads, catalysed esterification and transesterification in n-hexane under both batch and continuous-flow conditions. The operational stability of the lipase was markedly enhanced by alginate entrapment. In the esterification of butanoic acid with n-butanol, better results were obtained in the typical hydrophilic calcium alginate beads than in less hydrophilic matrices. The effects of substrate concentration, matrix area, and polarity of the substrate alcohols and of the organic solvent on the esterification activity were examined. The transesterification of octyl 2-bromopropanoate with ethanol was less efficient than that of ethyl 2-bromopropanoate with octanol. By using the hydrophilic alginate gel as an immobilization matrix in combination with a mobile hydrophobic phase, a two-phase liquid system was achieved with definite advantages for a continuous, enzyme-catalysed process.