Immobilized Candida antarctica lipase B on ZnO nanowires/macroporous silica composites for catalyzing chiral resolution of (R,S)-2-octanol

ZnO nanowires were successfully introduced into a macroporous SiO₂ by in situ hydrothermal growth in 3D pores. The obtained composites were characterized by SEM and XRD, and used as supports to immobilize Candida antarctica lipase B (CALB) through adsorption. The high specific surface area (233 m²/g) and strong electrostatic interaction resulted that the average loading amount of the composite supports (196.8 mg/g) was 3–4 times of that of macroporous SiO₂ and approximate to that of a silica-based mesoporous material. Both adsorption capacity and the activity of the CALB immobilized on the composite supports almost kept unchanged as the samples were soaked in buffer solution for 48 h. The chiral resolution of 2-octanol was catalyzed by immobilized CALB. A maximum molar conversion of 49.1% was achieved with 99% enantiomeric excess of (R)-2-octanol acetate under the optimal condition: a reaction using 1.0 mol/L (R,S)-2-octanol, 2.0 mol/L vinyl acetate and 4.0 wt.% water content at 60 °C for 8 h. After fifteen recycles the immobilized lipase could retain 96.9% of relative activity and 93.8% of relative enantioselectivity.     

Characterization of the lipase immobilized on Mg–Al hydrotalcite for biodiesel

Immobilization of Saccharomyces cerevisiae lipase by physical adsorption on Mg–Al hydrotalcite with a Mg/Al molar ratio of 4.0 led to a markedly improved performance of the enzyme. The immobilized lipase retained activity over wider ranges of temperature and pH than those of the free lipase. The immobilized lipase retained more than 95% relative activity at 50 °C, while the free lipase retained about 88%. The kinetic constants of the immobilized and free lipases were also determined. The apparent activation energies (E_a) of the free and immobilized lipases were estimated to be 6.96 and 2.42 kJ mol^?1, while the apparent inactivation energies (E_d) of free and immobilized lipases were 6.51 and 6.27 kJ mol^?1, respectively. So the stability of the immobilized lipase was higher than that of free lipase. The water content of the oil must be kept below 2.0 wt% and free fatty acid content of the oil must be kept below 3.5 mg KOH g [oil]^?1 in order to get the best conversion. This immobilization method was found to be satisfactory to produce a stable and functioning biocatalyst which could maintain high reactivity for repeating 10 batches with ester conversion above 81.3%.     

Development of a monolith based immobilized lipase micro-reactor for biocatalytic reactions in a biphasic mobile system

This paper reports a simple method for producing macroporous silica-monoliths with controllable porosity that can be used for the immobilization of lipases to generate an active and stable micro-reactor for biocatalysis. A range of commercially available lipases has been examined using the hydrolysis reactions of 4-nitrophenyl butyrate in water–decane media. The kinetic studies performed have identified that a similar value for k_cat is obtained for the immobilized Candida antarctica lipase A (0.13 min⁻¹) and the free lipase in solution (0.12 min⁻¹) whilst the immobilized apparent Michaelis constant K_m (3.1 mM) is 12 times lower than the free lipase in solution (38 mM). A 96% conversion was obtained for the immobilized C. antarctica lipase A compared to only 23% conversion for the free lipase. The significant higher conversions obtained with the immobilized lipases were mainly attributed to the formation of a favourable biphasic system in the continuous flowing micro-reactor system, where a significant increase in the interfacial activation occurred. The immobilized C. antarctica lipase A on the monolith also exhibited improved stability, showing 64% conversion at 80 °C and 70% conversion after continuous running for 480 h, compared to 40 and 20% conversions under the same temperature and reaction time for the free lipase.     

Ethyl isovalerate synthesis using Candida rugosa lipase immobilized on silica nanoparticles prepared in nonionic reverse micelles

Uniform and monodispersed silica nanoparticles were synthesized with a mean diameter of 100 ± 20 nm as analyzed by Transmission Electron Microscopy (TEM). Glutaraldehyde was used as a coupling agent for efficient binding of the lipase onto the silica nanoparticles. For the hydrolysis of pNPP at pH 7.2, the activation energy within 25–40 °C for free and immobilized lipase was 7.8 and 1.25 KJ/mol, respectively. The V_max and K_m of immobilized lipase at 25 °C for pNPP hydrolysis were found to be 212 μmol/min/mg and 0.3 mM, whereas those for free lipase were 26.17 μmol/min and 1.427 mM, respectively. The lower activation energy of immobilized lipase in comparison to free lipase suggests a change in conformation of the enzyme leading to a requirement for lower energy on the surface of the nanoparticles. A better yield (7 fold higher) of ethyl isovalerate was observed using lipase immobilized onto silica nanoparticles in comparison to free lipase.     

Synthesis of geranyl acetate in non-aqueous media using immobilized Pseudomonas cepacia lipase on biodegradable polymer film: Kinetic modelling and chain length effect study

Pseudomonas cepacia lipase (PCL) was immobilized on ternary blend biodegradable polymer made up of polylactic acid (PLA), chitosan (CH), and polyvinyl alcohol (PVA). Immobilized biocatalyst was characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), % water content, protein and lipase activity assay. The lipase activity assay showed enhanced activity of immobilized lipase than crude lipase. Higher half life time (t_1/2) and lower deactivation rate constant (K_d) was found for the n-hexane among various tested solvent. Influence of various reaction parameters on enzyme activity were studied in detail. When geraniol (1 mmol) and vinyl acetate (4 mmol) in toluene (3 mL) were reacted with 50 mg immobilized lipase at 55 °C; then 99% geraniol was converted to geranyl acetate after 3 h. Various kinetic parameters such as r_max, K_i(A), K_m(A), K_m(B) were determined using non-linear regression analysis for ternary-complex and Bi–Bi ping-pong mechanism. The kinetic study showed that reaction followed ternary-complex mechanism with inhibition by geraniol. Activation energy (Ea) was found to be lower for immobilized lipase (13.76 kCal/mol) than crude lipase (19.9 kCal/mol) indicating better catalytic efficiency of immobilized lipase. Immobilized biocatalyst demonstrated 4 fold increased catalytic activity than crude lipase and recycled five times.     

Optimization of immobilization conditions of Thermomyces lanuginosus lipase on styrene–divinylbenzene copolymer using response surface methodology

Microbial lipase from Thermomyces lanuginosus (formerly Humicola lanuginosa) was immobilized by covalent binding on a novel microporous styrene–divinylbenzene polyglutaraldehyde copolymer (STY–DVB–PGA). The response surface methodology (RSM) was used to optimize the conditions for the maximum activity and to understand the significance and interaction of the factors affecting the specific activity of immobilized lipase. The central composite design was employed to evaluate the effects of enzyme concentration (4–16%, v/v), pH (6.0–8.0), buffer concentration (20–100 mM) and immobilization time (8–40 h) on the specific activity. The results indicated that enzyme concentration, pH and buffer concentration were the significant factors on the specific activity of immobilized lipase and quadratic polynomial equation was obtained for specific activity. The predicted specific activity was 8.78 μmol p-NP/mg enzyme min under the optimal conditions and the subsequent verification experiment with the specific activity of 8.41 μmol p-NP/mg enzyme min confirmed the validity of the predicted model. The lipase loading capacity was obtained as 5.71 mg/g support at the optimum conditions. Operational stability was determined with immobilized lipase and it indicated that a small enzyme deactivation (12%) occurred after being used repeatedly for 10 consecutive batches with each of 24 h. The effect of methanol and tert-butanol on the specific activity of immobilized lipase was investigated. The immobilized lipase was almost stable in tert-butanol (92%) whereas it lost most of its activity in methanol (80%) after 15 min incubation.     

Enzymatic synthesis of fatty acid methyl esters from lard with immobilized Candida sp. 99-125

In this paper, immobilized lipase catalyzed biodiesel production from lard was studied. Using Candida sp. 99-125, the effect of temperature, water content, enzyme amount, solvent and three-step methanolysis were investigated. The optimal conditions for processing 1 g of lard were: 0.2 g immobilized lipase, 8 ml n-hexane as solvent, 20% water based on the fat weight, temperature 40 °C, and three-step addition of methanol. As a result, the fatty acid methyl esters (FAMEs) yield was 87.4%. The lipase was proved to be stable when used repeatedly for 180 h.     

Lipase immobilized microstructured fiber based flow-through microreactor for facile lipid transformations

A facile continuous flow-through Candida antarctica lipase B immobilized silica microstructured optical fiber (SMOF) microreactor for application in lipid transformations has been demonstrated herewith. The lipase was immobilized on the amino activated silica fiber using glutaraldehyde as a bifunctional reagent. The immobilized lipase activity in the SMOF was tested calorimetrically by determination of p-nitrophenyl butyrate hydrolysis products. The specific activity of the immobilized lipase was calculated to be 0.91 U/mg. The SMOF microreactor performance was evaluated by using it as a platform for synthesis of butyl laurate from lauric acid and n-butanol in n-hexane and n-heptane at 50 °C, with products identified by gas chromatography–mass spectrometry (GC–MS). Different substrate mole ratios were evaluated, with 1:3, lauric acid:n-butanol showing best performance. Remarkably, percentage yields of up to 99% were realized with less than ∼38 s microreactor residence time. In addition, the SMOF microreactor could be reused many times (at least 7 runs) with minimal reduction in the activity of the enzyme. The enzyme stability did not change even with storage of the microreactor in ambient conditions over one month.     

Optimization of Candida sp. 99-125 lipase catalyzed esterification for synthesis of monoglyceride and diglyceride in solvent-free system

Esterification of glycerol and oleic acid catalyzed by lipase Candida sp. 99-125 was carried out to synthesize monoglyceride (MAG) and diglyceride (DAG) in solvent-free system. Beta-cyclodextrin as an assistant was mixed with the lipase powder. Six reaction variables, initial water content (0–14 wt% of the substrate mass), the glycerol/oleic acid molar ratio (1:1–6:1), catalyst load (3–15 wt% of the substrate mass), reaction temperature (30–60 °C), agitator speed (130–250 r/min) and beta-cyclodextrin/lipase mass ratio (0–2) were optimized. The optimal conditions to the synthesis of MAG and DAG were different: the optimal glycerol/oleic acid molar ratio, beta-cyclodextrin/lipase mass ratio, catalyst load and reaction temperature were 6:1, 0, 5%, 50 °C for MAG, and 5:1, 1.5, 10%, 40 °C for DAG, respectively. The optimal water content and agitator speed for both MAG and DAG were 10% and 190 r/min, respectively. Under the optimal conditions, 49.6% MAG and 54.3% DAG were obtained after 8 h and 4 h, respectively, and the maximum of 81.4% MAG plus DAG (28.1% MAG and 53.3% DAG) was obtained after 2 h under the DAG optimal condition. Above 90% purity of MAG and DAG can be obtained by silica column separation.     

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10³, 98 × 10³, 147 × 10³ and 196 × 10³ U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters (K_m and V_max) were found using Michaelis–Menten enzyme kinetics. The K_m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.     

Hen egg white as a feeder protein for lipase immobilization

Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K_m (2.08 mM) and apparent V_max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K_m (8.0 mM) and V_max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.     

Efficient production of S-(+)-2-chlorophenylglycine by immobilized penicillin G acylase in a recirculating packed bed reactor

(S)-(+)-2-Chlorophenylglycine 1 is an important intermediate in the synthesis of Clopidogrel. A recirculating packed bed reactor (RPBR) was constructed for efficient production of (S)-1 by kinetic resolution of racemic N-phenylacetyl-2- chlorophenylglycine 2 using immobilized penicillin G acylase (PGA). The immobilized PGA exhibited maximum activity at 50 °C and pH 8.0 with (R,S)-2 as substrate. The kinetic constants (K_m and v_max) of immobilized PGA were calculated to be 20.61 mM and 83.2 mM/min/g, respectively. The substrate displayed inhibitory effect on immobilized PGA with inhibition constant of 221.23 mM. The immobilized PGA showed a strict enantiospecificity for substrate at different temperature, pH and substrate concentration examined. The performance and productivity of RPBR were evaluated by several critical parameters, including immobilized PGA load, substrate feeding rate, height to diameter ratio and so on. The kinetic resolution process shows higher initial reaction rate and conversion by recycling 100 mL of substrate solution (80 mM) through RPBRs packed with 6.0 g immobilized PGA with a feeding rate of 1.5 mL/min while the H/D ratio was 4.0. The immobilized PGA-catalyzed kinetic resolution of (R,S)-2 was successfully operated in the RPBR for 60 batches, with an average productivity of 1.2 g/L/h for (S)-1 in high optical purity (>97% enantiomeric excess) in semi-continuous operation. The residual (R)-2 can be easily racemized and then used as substrate.     

Impressive effect of immobilization conditions on the catalytic activity and enantioselectivity of Candida rugosa lipase toward S-Naproxen production

Candida rugosa lipase (CRL) was immobilized on Amberlite XAD 7 and the advantage of immobilization under the best reaction conditions in achieving high activity and enantioselectivity was shown for the hydrolysis of racemic Naproxen methyl ester. The performance of CRL was found to be better when the enzyme was immobilized at the temperature and pH values where higher conversion and enantioselectivity were obtained. The effects of immobilized lipase load, temperature, pH and substrate concentration on the conversion and enantioselectivity toward S-Naproxen production in aqueous phase/isooctane biphasic batch system were also evaluated. The increase in immobilized lipase load in 320–800 U/mL range increased the conversion of the substrate and enantioselectivity for S-Naproxen. The kinetic resolution of racemic Naproxen methyl ester conducted at the temperatures of 40, 45 and 50 °C and at the pH values of 4, 6, 7.5 and 9 resulted in the highest conversion and enantioselectivity at 45 °C and pH 6. Higher concentration of racemic Naproxen methyl ester than 10 mg/mL decreased both the conversion and enantioselectivity. CRL, which was immobilized at the temperature and pH values where the enzyme was more enantioselective, was successfully used in three successive batch runs each of 180 h. The highest enantiomeric ratio achieved in the S-Naproxen production was 174.2 with the conversion of 49%.     

Esterification activity and stability of Talaromyces thermophilus lipase immobilized onto chitosan

The Talaromyces thermophilus lipase (TTL) was immobilized by different methods namely adsorption, ionic binding and covalent coupling, using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the most suitable support material preserving the catalytic activity almost intact and offering maximum immobilization capacity (76% and 91%, respectively). The chitosan-immobilized lipase could be reputably used for ten cycles with more than 80% of its initial hydrolytic activity. Shift in the optimal temperature from 50 to 60 °C and in the pH from 9.5 to 10, were observed for the immobilized lipase when compared to the free enzyme.The catalytic esterification of oleic acid with 1-butanol has been carried out using hexane as organic solvent. A high performance synthesis of 1-butyl oleate was obtained (95% of conversion yield) at 60 °C with a molar ratio of 1:1 oleic acid to butanol and using 100 U (0.2 g) of immobilized lipase. The esterification product is analysed by GC/MS to confirm the conversion percentage calculated by titration.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

Enhancing performance of lipase immobilized on methyl-modified silica aerogels at the adsorption and catalysis processes: Effect of cosolvents

Hydrophobic silica aerogels modified with methyl group were applied as support to immobilize Candida rugosa lipase (CRL). At the adsorption process, different alcohols were used to intensify the immobilization of CRL. The results showed that n-butanol wetting the hydrophobic support prior to contacting with enzyme solution could promote lipase activity, but the adsorption quantity onto the support decreased. Based on this, a novel immobilization method was proposed: the support contacted with enzyme solution without any alcohols, and then the immobilized enzymes were activated by 90% (V) n-butanol solution. The experimental results showed that this method could keep high adsorption quantity (413.0 mg protein/g support) and increase the lipase specific activity by more than 50%. To improve the stability of immobilized lipase, the support after adsorption was contacted with n-octane to form an oil layer covering the immobilized lipases, thus the leakage can be decreased from over 30–4% within 24 h. By utilizing proper cosolvents, a high enzyme activity and loading capacity as well as little loss of lipase was achieved without covalent linkage between the lipase and the support. This is known to be an excellent result for immobilization achieved by physical adsorption only.     

A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: Characterization and application to enzymatic biodiesel production

To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40–50 °C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50 °C and was maintained even after an incubation of 24-h at 60 °C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50 °C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production.     

Baeyer–Villiger oxidation with peracid generated in situ by CaLB-CLEA catalyzed perhydrolysis

Candida antarctica lipase B, immobilized as cross linked enzyme aggregates (CLEAs) was used to mediate the Baeyer–Villiger oxidation of cyclohexanone to ɛ-caprolactone, and the reaction was compared with the one using Novozym^® 435 as catalyst. The conversion was dependent on the initial concentration of cyclohexanone, and was about 90% after 48 h at concentrations of up to 0.25 M but was decreased at higher concentrations. Caprolactone concentrations up to 0.6 M had no effect on the reaction efficiency. Among the cyclic ketones tested, the highest degree of conversion was achieved for cyclopentanone (88%) and the lowest for cyclooctanone (about 2%). The effect of methyl substitution and position of substitution on the cycloketone was studied using methylcyclohexanone and it has shown to influence the conversion efficiency. Both hydrogen peroxide and the reaction by-product acetic acid had a deleterious effect on the stability of the biocatalyst.     

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.     

Process intensification of immobilized lipase catalysis by microwave irradiation in the synthesis of 4-chloro-2-methylphenoxyacetic acid (MCPA) esters

4-Chloro-2-methylphenoxyacetic acid (MCPA) is a selective systemic herbicide which is absorbed by leaves and roots. MCPA esters are preferred due to their low water solubility and environmental friendliness. Esterification of MCPA with n-butanol was investigated as a model reaction using immobilized enzymes under the influence of microwave irradiation. Different immobilized enzymes such as Novozym 435, Lipozyme TL IM, Lipozyme RM IM and Lipase AYS Amano were studied under microwave irradiation amongst which Novozym 435 (immobilized Candida antarctica lipase B) was the best catalyst. Effects of various parameters were systematically studied on rates and conversion. Under microwave irradiation, the initial rates were observed to increase up to 2-fold. Under optimized conditions of 0.1 mmol MCPA and 0.3 mmol n-butanol in 15 mL 1,4-dioxane as solvent, Novozym 435 showed a conversion of 83% at 60 °C in 6 h. Based on initial rate and progress curve data, the reaction was shown to follow the Ping Pong bi–bi mechanism with inhibition by MCPA and n-butanol. Esterification of MCPA was also studied with different alcohols such as isopropyl alcohol, n-pentanol, n-hexanol, benzyl alcohol and 2-ethyl-1-hexanol.