Reaction Rates in Organic Media Show Similar Dependence on Water Activity with Lipase Catalyst Immobilized on Different Supports

Lipase (E.C. 3.1.1.3) from Rhizomucor miehei was adsorbed on silica, zirconia and five alumina support materials. The immobilised preparations were used to catalyse esterincation reactions of decanoic acid and dodecanol in hexane. The immobilised lipase and the organic phase were separately preequilibrated to the desired water activities. The various support materials adsorbed widely different amounts of water at a given water activity. The reaction rates with all the support materials show similar dependence on water activity when the rates were normalised with the optimal rate for that support material. Hence water activity predicts the optimal conditions much better than water content.     

Preparative scale enantioseparation of flurbiprofen by lipase-catalysed reaction

Effects of reaction media, alcohols and water on the enzyme activity of the immobilised Candida antarctica lipase were investigated for the separation of racemic flurbiprofen by an esterification reaction catalysed by immobilised enzyme in organic media. The S-enantiomer of flurbiprofen was directly resolved by the immobilised lipase esterification reaction in acetonitrile. Ping-Pong Bi–Bi kinetics were found to fit the initial reaction well of all the experimental runs. Model parameters for the reaction kinetics were evaluated from experiments at relatively low substrate concentrations, have shown to be applicable for preparative separation scale at high concentrations. Finally, the gram-scale production of single enantiomer with the optical purity of 93% e.e. was obtained.     

Covalent coupling method for lipase immobilization on controlled pore silica in the presence of nonenzymatic proteins

Candida rugosa lipase was covalently immobilized on silanized controlled pore silica previously activated with glutaraldehyde in the presence of nonenzymatic proteins. This strategy is suggested to protect the enzyme from aggregation effects or denaturation that occurs as a result of the presence of silane precursors used in the formation of the silica matrix. The immobilization yield was evaluated as a function of the lipase loading and the additive type (albumin and lecithin) using statistical concepts. In agreement with the mathematical model, the maximum coupling yield (32.2%) can be achieved working at high lipase loading (450 units x g(-1) support) using albumin as an additive. In these conditions, the resulting immobilized lipase exhibits high hydrolytic (153.2 U x mg(-1)) and esterification (337.6 mmol x g(-1) x min) activities. The enhanced activity of the final lipase derivative is the sum of the benefits of the immobilization (that prevents enzyme aggregation) and the lipase coating by additives that increases the accessibility of active sites to the substrate.     

Immobilisation of Candida rugosa lipase on polyhydroxybutyrate via a combination of adsorption and cross-linking agents to enhance acylglycerol production

In the present study, a combination of immobilisation processes was utilised to prepare robust biocatalysts. First, lipase from Candida rugosa was adsorbed on polyhydroxybutyrate (PHB) particles, followed by cross-linking with glutaraldehyde. Conditions for creating immobilised lipase involved the addition of 0.6 M glutaraldehyde and 45 U mL⁻¹ lipase while mixing at 150 rpm (4 °C) for 30 min. These conditions produced the highest yield of immobilised lipase (92 %) and the highest levels of activity (1.94 mg g⁻¹ support). At 40 °C and pH 9 the immobilised enzyme was optimally active with a K_m and V_maxat 1.2 mM and 2.5 × 10^-3 mmol min⁻¹, respectively. The use of immobilised lipase improved thermal stability, storage stability, and reusability.The immobilised lipase retained 80 % of its activity after incubation at 30–60 °C for 2 h and 4 °C for 30 d in 0.2 M sodium phosphate buffer (pH 7.0). Moreover, the immobilised enzyme retained 50 % of its activity after more than 14 cycles under optimal conditions. The immobilised lipase was used to produce monoacylglycerol MAG. The existence of a carbonyl group at 1,743 and 1,744 cm⁻¹ was identified using attenuated total reflectance (ATR)-Fourier transformed infrared spectroscopy. Results showed that 48 % MAG was produced.     

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.     

Lipase-catalysed synthesis of ester oils from biodiesel by-products

Ester oils obtained from natural long-chain fatty acids and alcohols are versatile substitutes for many petroleum-based products. Their efficient synthesis with the solvent-free esterification of free fatty acids (FFA) from by-products of biodiesel fabrication and 2-ethyl-1-hexanol with immobilised lipase from Thermomyces lanuginosa was investigated. The immobilisation of the biocatalyst in static emulsion yielded a specific esterification activity that was higher by a factor of 4.9–9.4 than the activity of the native enzyme. Favourable properties of the silicone-based immobilisation matrix in terms of stability and immobilisation yield were observed. In biodiesel by-products, the immobilised lipase catalysed the esterification of FFA as well as the transesterification of residual fatty acid methyl esters (FAME) to the desired ester oils. A conversion of 90% FFA and 35% FAME gave a total yield of 60%. The inactivation coefficients during repeated use in a stirred-tank reactor with intermittent pressure reduction were exceptionally low.     

Effects of water and silica gel on enzyme agglomeration in organic solvents

It has been observed that water, which is absolutely essential for enzyme activity, can induce the agglomeration of enzyme particles in organic media. Although enzyme agglomeration is significant in that it usually reduces enzyme activity and stability, little attention has been paid to the quantitative analysis of enzyme agglomeration behavior in nonaqueous bioactalytic systems. In this study, the effects of water and silica gel on enzyme agglomeration were investigated usingCandida rugosa lipase and cyclohexane as a model enzyme and an organic medium. The extent of enzyme agglomeration was quantified by sieve analysis of freeze-dried agglomerates. Increasing the water content of the medium increased the size of the enzyme agglomerates, and it was found that water produced during the esterification reaction could also promote the agglomeration of enzyme particles suspended in organic media. On the other hand, the size of the enzyme agglomerates was remarkably reduced in the presence of silica gel at the same water content. We also show that this increase in the size of enzyme agglomerates results in lower reaction rates in organic solvents.     

Properties of free and immobilised lipase from Burkholderia cepacia in organic media

The purified lipase from Burkholderia cepacia was immobilised on a porous polypropylene support and its biocatalytic properties were compared with those of the free enzyme in organic media. For both lipase preparations, the rate of p-nitrophenyl ester hydrolysis in n-heptane was not restricted by mass transfer limitations. The immobilisation changed neither the temperature at which the reaction rate was maximal, nor the activation energy of the reaction. The enzyme stability was slightly decreased (1.3-fold) upon immobilisation. Moreover, the immobilised enzyme displayed fewer variations of activity with fatty acid chain length. Interestingly, for all the different p-nitrophenyl esters used, the immobilised enzyme was more active (from 5.8- to 18.9-fold) than the free enzyme. Therefore, it would be very useful to use B. cepacia lipase immobilised onto porous polypropylene for applications in organic media, as it displayed high activities on a larger range of substrates. Received: 8 February 1999 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

Lipase-catalysed synthesis of ester oils from biodiesel by-products

Ester oils obtained from natural long-chain fatty acids and alcohols are versatile substitutes for many petroleum-based products. Their efficient synthesis with the solvent-free esterification of free fatty acids (FFA) from by-products of biodiesel fabrication and 2-ethyl-1-hexanol with immobilised lipase from Thermomyces lanuginosa was investigated. The immobilisation of the biocatalyst in static emulsion yielded a specific esterification activity that was higher by a factor of 4.9-9.4 than the activity of the native enzyme. Favourable properties of the silicone-based immobilisation matrix in terms of stability and immobilisation yield were observed. In biodiesel by-products, the immobilised lipase catalysed the esterification of FFA as well as the transesterification of residual fatty acid methyl esters (FAME) to the desired ester oils. A conversion of 90% FFA and 35% FAME gave a total yield of 60%. The inactivation coefficients during repeated use in a stirred-tank reactor with intermittent pressure reduction were exceptionally low.     

Optimized enzymatic synthesis of methyl benzoate in organic medium. Operating conditions and impact of different factors on kinetics

Enzyme-catalyzed synthesis of methyl benzoate is reported. It is the first example of direct esterification of benzoic acid which provides good yields. The reaction was performed in a heterogeneous medium by Candida rugosa lipase powder suspended in a hexane/toluene mixture. The impact of some factors was examined. Benzoic acid does not inhibit the lipase until 100 mM. Above 90 mM, methanol inhibits the enzyme. This inhibition is partially eliminated by increasing benzoic acid concentration. Below 90 mM, methanol mainly interacts with the water adsorbed on the biocatalyst. A minimum water content is necessary to activate the biocatalyst. Water must be provided proportionally to the lipase content. Toluene, necessary for benzoic acid solubilization, also acts negatively on reaction kinetics. This is attributed to a modification of benzoic acid partition between the biocatalytic and the organic phases. Copyright 1998 John Wiley & Sons, Inc.     

Growth Inhibition by Phenylalanine in Euglena gracilis

The hydrolysis and esterification by a thermostable lipase from Humicola lanuginosa No. 3 were investigated. Both reactions occurred readily at temperatures between 45~50°C. Esterification by the enzyme with glycerol was observed to be specific towards fatty acids with carbon numbers of C12~C18. Laurie acid esters with different alcohols such as primary alcohols, terpene alcohols, eie., were also synthesized readily. Esterification by the enzyme was adversely affected by the water content (optimum, ca. 7%), however, the hydrolysis rate increased rapidly with increasing water content (optimum, az. 60%). The enzyme showed increased activity in organic solvent-aqueous reaction systems. Nevertheless, hydrolysis in complete organic phase reactions was found not to be feasible. Hydrolysis at a higher temperature (50 or 55°C) in a solvent free phase was almost the same as that in organic solvent-aqueous phase reactions. The components of glycerides varied considerably during hydrolysis, whereby esterification resulted in a higher quantity of mono- and diglycerides (about 40%), compared to in the case of hydrolysis, for which the value was about 10~20%.     

Synthesis of 2-ethyl hexanol fatty acid esters in a packed bed bioreactor using a lipase immobilized on a textile membrane

An enzymatic process using a packed bed bioreactor with recirculation was developed for the scale-up synthesis of 2-ethylhexyl palmitate with a lipase from Candida sp. 99–125 immobilized on a fabric membrane by natural attachment to the membrane surface. Esterification was effectively performed by circulating the reaction mixture between a packed bed column and a substrate container. A maximum esterification yield of 98% was obtained. Adding molecular sieves and drying the immobilized lipase both decreased the water content at the reactor outlet and around the enzyme, which led to an increase in the rate of esterification. The long-term stability of the reactor was tested by continuing the reaction for 30 batches (over 300 h) with an average esterification yield of about 95%. This immobilized lipase bioreactor is scalable and is thus suitable for industrial production of 2-ethylhexyl palmitate.     

Lipase-catalyzed esterification of hydrophilic diols in organic solvents

Summary The direct, lipase-catalyzed esterification of hydrophilic diols in organic solvents was achieved by first adsorbing the hydrophilic, solvent immiscible substrate onto a solid support with high internal surface, namely silica gel and reacting the solid mixture with fatty acid vinyl esters in an appropriate organic solvent and in presence of an immobilized lipase fromMucor miehei (Lipozyme). Quantitative conversions of the acyl donors and very high reaction rates were observed in these transformations. Furthermore, mono- or diesters of these diols could be selectively produced by this method.     

Porous zirconia: a new support material for enzyme immobilization

Four different proteases (trypsin, chymotrypsin, papain and pepsin) were covalently attached to the surface of a new type of porous zirconia, as well as a conventional porous silica, activated with 3-isothiocyanatopropyltriethoxy silane (NCS-silane). The immobilization efficiency onto the porous zirconia material was evaluated in terms of the amount of enzyme attached to the particles and from the biological activity remaining after the immobilization step. The results were compared with the corresponding experiments with a porous silica of similar surface area/g support material. In addition, the storage stability of the modified zirconia and silica biocatalysts were evaluated. These results indicated that specific immobilized enzyme biocatalysts can be achieved with this new zirconia support material which exhibits different properties to those observed with the more conventional silica-based materials. Moreover, the results with the enzyme-zirconia biocatalysts also indicate different characteristics when compared with data for the same enzymes immobilized under similar buffer conditions to organic support materials as previously described by various other investigators. The advantages of zirconia-based immobilized enzyme biocatalysts in terms of their density and chemical robustness are also described relative to other alternative support materials currently in use.     

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.     

Orientation and conformation of a lipase at an interface studied by molecular dynamics simulations

Electron density profiles calculated from molecular dynamics trajectories are used to deduce the orientation and conformation of Thermomyces lanuginosa lipase and a mutant adsorbed at an air-water interface. It is demonstrated that the profiles display distinct fine structures, which uniquely characterize enzyme orientation and conformation. The density profiles are, on the nanosecond timescale, determined by the average enzyme conformation. We outline a computational scheme that from a single molecular dynamics trajectory allows for extraction of electron density profiles referring to different orientations of the lipase relative to an implicit interface. Profiles calculated for the inactive and active conformations of the lipase are compared with experimental electron density profiles measured by x-ray reflectivity for the lipase adsorbed at an air-water interface. The experimental profiles contain less fine structural information than the calculated profiles because the resolution of the experiment is limited by the intrinsic surface roughness of water. Least squares fits of the calculated profiles to the experimental profiles provide areas per adsorbed enzyme and suggest that Thermomyces lanuginosa lipase adsorbs to the air-water interface in a semiopen conformation with the lid oriented away from the interface.     

Effect of serum concentration on retroviral vector production from the amphotropic ψCRIP murine producer cells

The production of ethanol by Saccharomyces cerevisiae immobilized cells and its esterification with oleic acid, catalysed by a lipase from Rhizomucor miehei, was the biochemical process considered as model to illustrate the concept of extractive biocatalysis. The selection of the most suitable support for lipase immobilization was carried out. The best results for the ethanol/oleic acid esterification reaction were obtained with the lipase adsorbed on a polyamide type support, Accurel EP 700. The immobilization method was optimized in terms of immobilization pH, contact time and protein/support ratio. The better performances of the extractive fermentations of ethanol were obtained when entrapped k-carrageenan Saccharomyces cerevisiae cells and a lipase from Rhizomucor miehei, free or immobilized in Accurel EP 700, were used simultaneously. The observed reutilization capacity of the immobilized enzyme could be advantageous for its application in a continuous reactor.     

Lipase immobilisation: an equilibrium study of lipases immobilised on hydrophobic and hydrophilic/hydrophobic supports

This work investigates the enzyme-support equilibrium behaviour in immobilised lipase biocatalysts. Equilibrium data determines the maximum enzyme up-take by unit weight of support. Four lipases were immobilised on two polymeric supports, respectively. They were Lipase PS from Pseudomonas, Lipolase 100L from Humicola, SP871 from Rhizomucor miehel and QL from Alcaligenes. The supports were Accurel EP100 (a polypropylene material) and 45SAA (a polypropylene/silica composite). Experimentally, equilibrium was expressed in terms of lipase loading (LU/g support) versus residual lipase concentration (LU/dm³). Activity, efficiency and operational stability of the immobilised lipases were assayed by solvent-free esterification of oleic acid and octanol.Equilibrium data were modelled by the Langmuir, Freundlich and Redlich–Peterson formulae. It was found that Lipolase 100L/Accurel, PS/45SAA and SP871/45SAA systems conformed to the Langmuir behaviour, while Lipase PS/Accurel and SP871/Accurel systems followed the Freundlich behaviour and Lipolase 100L/45SAA, QL/45SAA and QL/Accurel EP100 resembled Redlich–Peterson behaviour. Whereas immobilisation on Accurel EP100 resulted in classical equilibrium isotherms with all four lipases, immobilisation on support 45SAA resulted in two-plateau equilibrium curves which included a step change in the isotherm for all lipases studied, except for SP871. Quantitatively, for 1 g lipase, Accurel and 45SAA had a maximum capacity of 140 and 260 kLU for PS, 112 and 550 kLU for Lipolase 100L, 320 and 800 kLU for SP871 and 18 and 29 kLU for QL, respectively.     

Water activity and substrate concentration effects on lipase activity

Catalytic activity of lipases (from Rhizopus arrhizus, Canadida rugosa, and Pseudomonas sp. was studied in organic media, mainly diisopropyl ether. The effect of water activity (a(w)) on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes. This was shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase. In the esterification reaction the K(m) for the acid substrate showed a slight increase with increasing water activities. On the other hand, the K(m) for the alcohol substrate increased 10-20-fold with increasing water activity. The relative changes in K(m) were shown to be independent of the enzyme studied and solvent used. The effect was attributed to the increasing competition of water as a nucleophile for the acyl-enzyme at higher water activities. In a hydrolysis reaction the K(m) for the ester was also shown to increase as the water activity increased. The effect of water in this case was due to the fact that increased concentration of one substrate (water), and thereby increased saturation of the enzyme, will increase the apparent K(m) of the substrate (ester) to be determined. This explained why the hydrolysis rate decreased with increasing water activity at a fixed, low ester concentration. The apparent V(max) for R. arrhizus lipase was similar in four of six different solvents that were tested; exceptions were toulene and trichloroethylene, which showed lower values. The apparent K(m) for the alcohol in the solvents correlated with the hydrophobicity of the solvent, hydrophobic solvents giving lower apparent K(m). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 798-806, 1997.     

Application of advanced materials as support for immobilisation of lipase from Candida rugosa

Ni/Al-layered double hydroxides (Ni-LDHs) and Ni/Al-sodium dodecyl sulfonate layered double hydroxide nanocomposites (Ni-SDS-LDHs) with a molar ratio of Ni:Al (4:1) have been prepared by a co-precipitation (or salt-base) method. Their structures were determined using Powder X-Ray Diffractometer (PXRD) and the spectra showed that basal spacings for Ni-LDHs and Ni-SDS-LDHs synthesised were around 8.1?Å and 34.8?Å, respectively. Lipase from Candida rugosa was immobilised onto these advanced materials, by physical adsorption. The activity of immobilised lipase was investigated through esterification of palmitic acid and isopropyl alcohol in hexane. The effects of reaction temperature, thermostability, stability in organic solvent, operational stability, leaching and storage studies of the immobilised lipase were investigated. These biocatalysts exhibited higher activities than the native lipase with an optimum temperature of 40°C. Immobilised lipases showed higher storage stability than native lipase (up to 60 days) and during operational studies at 30°C for 5?h, more than 50% of its activity was retained. Leaching studies showed that physical adsorption is suitable for the attachment of enzymes onto LDHs.