Lipase‐catalysed esterification of oleic acid and ethanol in a continuous packed bed reactor,using supercritical CO2 as solvent: approximation of system kinetics

This work investigated the immobilised lipase kinetics of esterification of oleic acid and ethanol. The reaction was conducted under supercritical conditions (13 × 10⁶ Pa and 40 °C) using carbon dioxide as solvent in a continuous packed bed (plug flow) reactor. Biocatalyst Lypozyme^TM IM60, which is lipase from Rhizomucor miehei (EC.3.1.1.3), immobilised on Duolite (anionic exchange resin) was used as biocatalyst. Kinetically, with regard to oleic acid, the reaction was successfully modelled by the Michaelis–Menten mechanism. The reaction rate constants K_m and V_max were evaluated. Furthermore, it was found to undergo competitive inhibition by ethanol, and the inhibition constant K_i was evaluated. © 2000 Society of Chemical Industry     

Production of structured triacylglycerols in an immobilised lipase packed‐bed reactor: batch mode operation

Structured triacylglycerols with caprylic acid at the sn‐1 and sn‐3 positions of the glycerol backbone and eicosapentaenoic acid (EPA) at the position sn‐2 were synthesised by acidolysis of a commercially available EPA‐rich oil (EPAX4510, Pronova Biocare) and caprylic acid catalysed by the 1,3‐specific immobilised lipase Lipozyme IM. The reaction was carried out in an immobilised lipase packed‐bed reactor by recirculating the reaction mixture through the bed. The exchange equilibrium constants between caprylic acid and the native fatty acids of EPAX4510 were determined. The n‐3 polyunsaturated fatty acids (PUFAs), EPA and docosohexaenoic acid (DHA), were the most easily displaced by the caprylic acid. The exchange equilibrium constants were 3.68 and 3.06 for EPA and DHA, respectively. The influence of the flow rate of the reaction mixture through the packed‐bed and the substrate concentration in the reaction rate were studied. For flow rates between 74 and 196 cm³ h^?1 (bed of 6.6 mm internal diameter and 0.46 porosity) and triacylglycerol concentrations between 0.036 and 0.108 M , the data fitted well to an empirical kinetic model which allowed representative values of the apparent kinetic constant to be obtained. Hence, the average reaction rates and kinetic constants of exchange of caprylic acid and native fatty acids of EPAX4510 could be calculated. In the conditions indicated, the parameter (lipase mass × time/triacylglycerol mass, m_Lt/V[TG]₀) constituted the intensive variable of the process for use in predicting the composition of structured triacylglycerols at different reaction times. At equilibrium, the structured triacylglycerol produced had the following composition: caprylic acid 59.5%, EPA 9.6%, DHA 2.2% and oleic acid 11.8%. Copyright © 2004 Society of Chemical Industry     

Poly(hydroxyethyl methacrylate‐co‐glycidyl methacrylate) reactive membrane utilised for cholesterol oxidase immobilisation

Poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) p(HEMA–GMA) membrane was prepared by UV‐initiated photopolymerisation of 2‐hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA) in the presence of an initiator, azobisisobutyronitrile (AIBN). Cholesterol oxidase was immobilised directly on the membrane by forming covalent bonds between its amino groups and the epoxide groups of the membrane. An average of 53 µg of enzyme was immobilised per cm² of membrane, and the bound enzyme retained about 67% of its initial activity. Immobilisation improved the pH stability of the enzyme as well as its temperature stability. The optimum temperature was 5 °C higher than that of the free enzyme and was significantly broader. The thermal inactivation rate constants for free and immobilised preparations at 70 °C were calculated as k_{i (free)} 1.06 × 10^?1 min^?1 and k_{i (imm)} 2.68 × 10^?2 min^?1, respectively. The immobilised enzyme activity was found to be quite stable in the repeated experiments. © 2002 Society of Chemical Industry     

One‐Pot Lipase Entrapment Within Silica Particles to Prepare a Stable and Reusable Biocatalyst for Transesterification

In order to enhance the reusability, Rhizomucor miehei lipase was entrapped in a single step within silica particles having an oleic acid core (RML@SiO₂). Characterization of RML@SiO₂ by scanning and transmission electron microscopy and Fourier transform infrared studies supported the lipase immobilization within silica particles. The immobilized enzyme was employed for transesterification of cottonseed oil with methanol and ethanol. Under the optimum reaction conditions of a methanol‐to‐oil molar ratio of 12:1 or ethanol‐to‐oil molar ratio of 15:1, stirring speed of 250 revolutions/min (flask radius = 3 cm), reaction temperature of 40 °C, and biocatalyst concentration of 5 wt% (with respect to oil), more than 98 % alkyl ester yield was achieved in 16 and 24 h of reaction duration in case of methanolysis and ethanolysis, respectively. The immobilized enzyme did not require any buffer solution or organic solvent for optimum activity; hence, the produced biodiesel and glycerol were free from metal ion or organic molecule contamination. The activation energies for the immobilized enzyme‐catalyzed ethanolysis and methanolysis were found to be 34.9 ± 1.6 and 19.7 ± 1.8 kJ mol^?1, respectively. The immobilized enzyme was recovered from the reaction mixture and reused in 12 successive runs without significant loss of activity. Additionally, RML@SiO₂ demonstrated better reusability as well as stability in comparison to the native enzyme as the former did not lose the activity even upon storage at room temperature (25–30 °C) over an 8‐month period.     

Kinetic studies of Mucor miehei lipase in phosphatidylcholine microemulsions

Ethanol—oleic acid esterification by a free and microencapsulated lipase from Mucor miehei, using dodecane as solvent and phosphatidylcholine as surfactant, was studied. The initial esterification rate was influenced by the water content in the biphasic system. Kinetic studies with free and microencapsulated enzyme showed that the microencapsulation led to an increase of the kinetic parameters (V_max and K_m), probably due to an increase of the interfacial area. The reaction rate was also affected by the shaking rate, the temperature and the pH. The optimal temperature and pH achieved were, respectively, 40°C and 4.5 using free enzyme, and 50°C and 6 using microencapsulated enzyme.     

Kinetics of lipase‐catalysed interesterification of triolein and caprylic acid to produce structured lipids

The influence of the molar ratio caprylic acid/triolein, enzyme concentration and water content on the kinetics of the interesterification reaction of triolein (TO) and caprylic acid (CA) were studied. The enzyme used was the 1,3‐specific Rhizomucor miehei lipase. Data modelling was based on a simple scheme in which the acid was only incorporated in positions 1 and 3 of the glyceride backbone. In addition, it was assumed that positions 1 and 3 of the triglycerides were equivalent and that the events at position 1 did not depend on the nature of the fatty acid in position 3 and vice versa. Monoglycerides and diglycerides were not detected during the experiments. This was attributed to the low water content of the immobilised enzyme particles. The value of the equilibrium constant, K, for the exchange of caprylic and oleic acids was 2.7, which indicated that the incorporation of caprylic acid into triglycerides was favoured compared with the incorporation of oleic acid. Simple first order kinetics could describe the interesterification reaction. Using this model and the calculated equilibrium constant, the apparent kinetic constants were calculated. The model fitted all the experimental data except for the CA/TO molar ratios larger than 6. Moreover, the interesterification reaction rate had a maximum value at CA/TO molar ratios of 4–6 mol mol^?1. Copyright © 2003 Society of Chemical Industry     

Protection by different agents against inactivation of lipoxygenase by hydrogen peroxide

H₂O₂ is a potent inactivator of lipoxygenase. In his paper, the ability of different agents [mannitol, oleic, stearic and linoleic acid,n-butanol, and hydroperoxy octadecadienoic acid (HPOD)] to prevent the inactivation of tomato lipoxygenase by hydrogen peroxide has been studied. The involvement of OH^· in the inactivation process is suggested by the ability of mannitol to prevent the loss of activity. This radical would be produced by reaction of H₂O₂ with the Fe(II) lipoxygenase. The most effective protection was displayed by HPOD, the product of the reaction of lipoxygenase with linoleic acid. This result could be explained by the conversion of the native enzyme into the Fe(III) lipoxygenase in the presence of HPOD; the Fe(III) enzyme is not able to react with H₂O₂, and no OH^· will be produced. The protective effect obtained with oleic and stearic acid could be explained by an occupation of the active center by these inhibitors. The enzyme would not transform them, but their presence would hamper the conversion of H₂O₂ in OH^· and limit the damage in the active center.     

Electron Transfer and Stability of a Quinohaemoprotein Alcohol Dehydrogenase Electrode

Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni (QH-EDH) was immobilised in a redox polymer network of a polyvinylpyridine, partially N-complexed with osmiumbis(bipyridine)chloride. Substrate-dependent electron transfer occurred, indicating that the enzyme was active and that effective electron transport was achieved. It was shown that the enzyme molecular weight is of importance with respect to the enzyme electrode stability. Long term stability and current density of the QH-EDH electrodes were highest when the enzyme was immobilised at pH 10·0 and 4°C, followed by an additional cross-linking step with glutaraldehyde (1%) at pH 7·0. With such an electrode current densities of 40 μA cm⁻² were obtained for several primary alcohols. The affinity of the immobilised enzyme for these substrates (K_m(app) values) was similar to that of the enzyme in solution. The half-life time of the electrodes was between 50 h and 200 h depending on the time the enzyme was in contact with the substrate. When the immobilised enzyme electrode was operated at temperatures above 37°C the stability decreased. © 1997 SCI.     

Kinetic studies on immobilised lipase esterification of oleic acid and octanol

The present work investigates the reaction kinetics of immobilised lipase esterification of oleic acid and octanol, in a solvent-free system. Lipase from Rhizomucor miehei was immobilised on a hydrophobic support. The initial reaction rate was investigated as a function of octanol concentration and temperature, and the reaction kinetics were described in terms of the Michaelis–Menten mechanism. Evaluating K_m, V_max and k_cat/K_m as a function of temperature, it was found that K_m was minimum and k_cat/K_m was maximum at 40°C while V_max was maximum at 50°C. Furthermore, applying the Ping Pong Bi Bi mechanism yielded good results for this two-substrate system.     

Enzymatic synthesis of oleyl oleate in dense fluids

Esterification between oleic acid and oleyl alcohol, catalyzed by theMucor miehei immobilized lipase in a batch-stirred tank reactor with supercritical carbon dioxide as solvent produced higher reaction rates at supercritical conditions than in the solvent-free system. A continuous fixed-bed reactor was designed based on the results obtained from batch experiments. At 150 bar, 40°C, and with water activity 0.46% w/w, the activity of the enzyme preparation is practically unchanged when CO₂ was used as solvent. The addition of small amounts of water increases the conversion rate. The higher conversion also was observed at longer residence time. Whenn-butane was used as reaction medium, a decrease in conversion was observed.     

Effect of pressure on the extractive biocatalysis of ethanol

The effect of pressure on the esterification reaction of ethanol with water-immiscible organic acids, catalysed by a lipase from Mucor miehei (pH 4.5; 30°C), was studied through analysis of the kinetics and equilibrium parameters. An increase of the ethanol distribution between the aqueous and organic phases was observed by the addition of lipase and the increase of the pressure in the system. Furthermore, the enzyme showed high specificity for the acid substrate, esterifying preferentially long chain fatty acids (C₈-C₁₈). In the studies described oleic acid was used as substrate for the esterification reaction. A kinetic study with the free enzyme, showed that pressure affected the extraction system, increasing the maximum reaction rate (> V_max), the affinity (< K_m) and the specificity (> V_max/K_m = k_sp) of the enzyme to the substrate, probably due to the effect of pressure on the electrostatic interactions in biological systems. The enzyme operational stability, at 30°C, improved significantly with the increase of pressure, having lower values for the deactivation constant (k) (8.3 × 10^?3 h^?1) and higher values for the half-life times (t_1/2) (77 h) in comparison with those obtained under atmospheric pressure conditions (k = 2.3 × 10^?2h^?1; t_1/2 = 30 h).     

Lipase‐catalyzed esterification of cinnamic acid and oleyl alcohol in organic solvent media

The esterification of cinnamic acid (CA) and oleyl alcohol (OA) in organic solvent media by immobilized lipase Novozym 435 was optimized in terms of selected parameters, including the logarithm of the 1‐octanol/water partition coefficient of the organic solvent (log P, 0.29–4.5), initial water activity (a_w, 0.05–0.75), agitation speed (0–200 rpm), temperature (35–65 °C) and ratio of substrates (CA/OA, 1.0:0.5–1.0:6.0). The results showed that the more hydrophobic solvent mixtures and lower initial a_w values resulted in a higher enzymatic activity and bioconversion yield. The most appropriate solvent medium and initial a_w value was the mixture of iso‐octane/2‐butanone (85:15, v/v) and 0.05, respectively. The results also showed that an agitation speed of 150 rpm and a reaction temperature of 55 °C were optimal for the reaction system. The activation energy (E_a) of the esterification reaction was calculated as 43.6 kJ mol^?1. The optimal ratio of CA to OA was 1.0:6.0, with the absence of any inhibition by OA. Using the optimized conditions, the maximum enzymatic activity was 390.3 nmol g^?1 min^?1, with a bioconversion yield of 100% after 12 days of reaction. In addition, the electrospray ionization‐mass spectroscopy analysis confirmed that the major end product of the esterification reaction was oleyl cinnamate. Copyright © 2005 Society of Chemical Industry     

Characterisation of acorn oils extracted by hexane and by supercritical carbon dioxide

Acorn fruit oils from two species of oak, Quercus rotundifolia L. (holm‐oak) and Quercus suber L. (cork‐oak), were extracted by n‐hexane. The acorn fruit of Quercus rotundifolia L. was also extracted by supercritical CO₂ at 18 MPa and 313 K, a superficial velocity of 2.5 × 10^?4 ms^?1, and a particle size diameter of 2.7 × 10^?4 m. The oils were characterised in terms of fatty acids, triglycerides, sterols, tocopherols, and phospholipids. The main fatty acid in both fruit species was oleic acid (about 65%), followed by linoleic acid (about 16.5–17%) and palmitic acid (about 12.1–13.4%). The main triglyceride found in acorn oils was the OOO (oleic, oleic, oleic) triglyceride (33–38%), followed by the POO (palmitic, oleic, oleic) triglyceride (12.6–18.2%). In terms of sterols, the main component in acorn oils of both species was β‐sitosterol (83.5–89%), followed by stigmasterol (about 3%). However, in Quercus suber L., acorn oil was found to consist to 10.2% of campesterol. The amount of cholesterol was low (0.27% for the Quercus rotundifolia L. oil extracted by supercritical fluid extraction, and 0.18% for the oil extracted by n‐hexane). The Quercus suber L. acorn oil presented 0.1% of cholesterol. The total amount of tocopherols in Quercus rotundifolia L. acorn oils was almost the same when the oil was extracted by n‐hexane (973 mg/kg oil) or by supercritical CO₂ (1006 mg/kg oil). The Quercus suber L. acorn oil presented a high value of total tocopherols (1486 mg/kg oil). The supercritical CO₂ did not extract the phospholipids. The amount of phospholipids was very similar for both species of oak acorn oils extracted by n‐hexane. Oxidative stability was also studied, by using the peroxide value and the Rancimat method, revealing that all the oils were significantly protected against oxidation. The influence of storage, under several conditions, on the oxidative stability was also studied. The Quercus rotundifolia L. oil extracted by n‐hexane was better protected against oxidation after a few days of storage at 60 °C.     

The preparation of an immobilised glucose isomerase II. Immobilisation and properties of the immobilised enzyme

Glucose isomerase ex Lactobacillus brevis was successfully immobilised on microcrystalline cellulose, using the transition metal-link method. Immobilisation could be performed over a pH range of 5 to 9, and usually resulted in an apparent specific activity increase. The immobilised glucose isomerase generally displayed properties similar to those of the soluble enzyme, with the exception of the following differences:

• (i) a pH optimum at pH = 6, an acid shift of 0.5 units on immobilisation;
• (ii) an optimum reaction temperature at 50 °C, lower than that for the soluble enzyme;
• (iii) on incubation at 4 °C, a retention of 53% of the initial specific activity, when stored in 0.02 M, pH = 7, Tris buffer, after 8 weeks, compared with an apparent activation of the soluble enzyme after 10 and 19 weeks' storage.

Storage properties of the immobilised enzyme at 4 °C in Tris were apparently improved by the presence of Mn⁺⁺ and Co⁺⁺, although associated with some protein release. Storage at 4 °C in water alone, as opposed to Tris, resulted in a more rapid activity loss.     

Lipase/acyltransferase‐catalysed interesterification of fat blends containing n‐3 polyunsaturated fatty acids

The lipase/acyltransferase from Candida parapsilosis is an original biocatalyst that preferentially catalyses alcoholysis over hydrolysis in biphasic aqueous/organic media. In this study, the performance of the immobilised biocatalyst in the interesterification in solvent‐free media of fat blends rich in n‐3 polyunsaturated fatty acids (n‐3 PUFA) was investigated. The interesterification activity of this biocatalyst at a water activity (a_w) of 0.97 was similar to that of commercial immobilised lipases at a_w values lower than 0.5. Thus, the biocatalyst was further used at an a_w of 0.97. Response surface modelling of interesterification was carried out as a function of medium formulation, reaction temperature (55–75 °C) and time (30–120 min). Reaction media were blends of palm stearin (PS), palm kernel oil and triacylglycerols (TAG) rich in n‐3 PUFA (“EPAX 4510TG”; EPAX AS, Norway). The best results in terms of decrease in solid fat content were observed for longer reaction time (>80 min), lower temperature (55–65 °C), higher “EPAX 4510TG” content and lower PS concentration. Reactions at higher temperature led to final interesterified fat blends with lower free fatty acid contents. TAG with high equivalent carbon number (ECN) were consumed while acylglycerols of lower ECN were produced.     

Lipase-catalyzed synthesis of chiral triglycerides

Under certain reaction conditions, the acidolysis of tripalmitin with oleic acid using immobilized lipase from Rhizomucor miehei resulted in a higher level of monosubstituted oleoyldipalmitoyl (OPP) triglycerides than had been predicted according to kinetic modeling. The reaction products were subjected to chiral analysis by high-performance liquid chromatography (HPLC), which indicated that the enzyme was more active at the sn-1 position of the triglyceride than at the sn-3 position, resulting in synthesis of the chiral triglyceride 1-oleoyl-2,3-dipalmitoyl-sn-glycerol. A kinetic model was developed and was correlated with the HPLC method to provide a simple means to predict the stereoselectivity of lipase-catalyzed reactions. By using the model, the stereoselectivity of immobilized Rhizomucor miehei lipase was found to depend strongly on the initial water activity (a _w) of the reaction mixture, with greater selectivity occurring at lower a _w. The sn-1 selectivity was essentially maintained using various solvents, or without solvent, when a _w was kept constantly low. Variation in the fatty acid composition of the triglyceride indicated that shorter-chain fatty acids result in greater stereoselectivity, while variation of the chainlength of the free fatty acid indicated an enhancement by the longest chainlength. The stereoselectivity of this lipase was confirmed using a new ¹³C nuclear magnetic resonance method. By using immobilized R. miehei lipase at low a _w approximately 80% of the chiral triglyceride found in the reaction mixture was the sn-1 enantiomer, at high reaction conversion.     

Enzymatic Synthesis of High sn-2 DHA and ARA Modified Oils for the Formulation of Infant Formula Fat Analogues

High sn‐2 docosahexaenoic and arachidonic acid oils (DHAO_m and ARAO_m, respectively) were produced independently via enzymatic interesterification of DHA‐rich and ARA‐rich single cell oils (DHASCO and ARASCO, respectively) using a mix of immobilized lipases, Lipozyme^® TL IM and Novozym^® 435 (weight ratio 1:1) as the biocatalyst system. Response surface methodology (RSM) was employed to model and optimize the reactions conditions. Three independent variables, namely reaction time, reaction temperature, and enzyme load, were investigated in DHAO_m and ARAO_m models. The prediction power of the model was further confirmed by solvent‐free scale‐up reactions of 100 g per batch. Final results showed that DHAO_m contained 46.53 mol% of total DHA (49.70 % at the sn‐2 position), while ARAO_m contained 47.25 mol% of total ARA (36.08 % at the sn‐2 position). This represents a significant increment in the amount of DHA and ARA at the sn‐2 position when compared to DHASCO (47.8 mol%; 30.30 % at the sn‐2) and ARASCO (47.79 mol%; 28.50 % at the sn‐2), respectively. These products have potential as additions to infant formulas where DHA and ARA supplementation is required.     

Parameter optimization for the enzymatic hydrolysis of sunflower oil in high-pressure reactors

This study is concerned with the hydrolysis of sunflower oil in the presence of lipase preparation Lipolase 100T (Aspergillus niger lipase). Supercritical carbon dioxide was used as a solvent for this reaction. In a high-pressure stirred tank reactor operated in a batch mode, the effects of various process parameters (temperature, pressure, enzyme/substrate ratio, pH, and oil/buffer ratio) were investigated to determine the optimal reaction rate and conversion for the hydrolysis process. The optimal concentration of lipase was 0.0714 g/mL of CO₂-free reaction mixture, and the highest conversions of oleic acid (0.193 g/g of oil phase) and linoleic acid (0.586 g/g of oil phase) were obtained at 50°C, 200 bar, pH=7, and an oil/buffer ratio of 1∶1 (w/w).     

Kinetics and reaction engineering of penicillin G hydrolysis

A general diffusion reaction model for immobilised biocatalysis has been developed. The model has been used to study the deacylation of penicillin G to 6-aminopenicillanic acid using two commercially available immobilised Penicillin acylases. The values of D_e/R² for the enzyme pellets have been estimated using data on uptake of 6-aminopenicillanic acid and phenylacetic acid by the enzyme pellets. The kinetic parameters of the model were individually estimated from a suitably designed set of experiments. The values of the Thiele modulus from the kinetic parameters so calculated have been found to be in the range 1·67 to 9·8 for the two enzymes studied, implying that diffusional effects cannot be ignored. The effect of such diffusional limitations on the overall rates and hence on the utilisation of the intrinsic kinetic ability of the enzyme has been demonstrated. This paper also reports on the implementation of the fed-batch strategy for this system. The proposed strategy, which involves maintaining the substrate concentration at the optimum value for a large part of the conversion, results in higher product concentrations than in batch operation, thereby reducing downstream procesing costs. The productivity was also shown to be considerably higher than for batch operation. Further, the ease of implementation of this mode of operation has been demonstrated.     

Preparation of bimodal mesoporous silica containing cerious salt and its application as catalyst for the synthesis of biodiesel by esterification

A kind of bimodal mesoporous silica catalyst modified with ammonium cerous sulfate (ACS/BMMS) was synthesized and applied in the esterification of free fatty acid and alcohol. The characterization results including XRD, N₂ adsorption and desorption, FTIR, ²⁹Si-NMR and TEM showed that ACS/BMMS has orderly arranged bimodal mesopores, the small mesopore diameter is about 4.0–6.0 nm and the large mesopore diameter is in the range of 7.0–9.0 nm. The chemical interaction existed between silica group Si (OH)₂(OSi)₂ and the NH₄ ⁺, SO₄ ^2? and Ce-O groups of cerious salt. When the loading is not more than 10%, cerious salt dispersed finely on the supports. Oleic acid and methanol were used as the raw material of probe reaction; ACS/BMMS had significantly better activity than the ACS/SBA-15, ACS/SBA-16, ACS/MCM-41, BMMS and bulk ACS. The optimum loading of ACS is 10%, the optimum reaction conditions are reaction temperature 140 °C, reaction time 2 h, mole ratio of methanol to oleic acid 2.0 and the dosage of catalyst 4.0%, in above situation the conversion of oleic acid is about 94.0%, the reusability of ACS/BMMS is much better than bulk ACS. The kinetic study showed that the esterification of oleic acid and methanol on ACS/BMMS match Eley–Rideal model very well.