Strategies for enzymatic esterification in organic solvents: comparison of microaqueous, biphasic, and micellar systems.

The kinetics of butyl butyrate synthesis by a lipase from Mucor miehei in different types of organic media were investigated. The three systems studied were a microaqueous medium containing enzyme in suspension in hexane, a water-hexane two-phase system, and reverse micelles. The synthesis of butyl butyrate was possible in all cases because of a favorable partition of the ester into the organic solvent. A sufficient stirring rate was necessary to achieve good reaction rates in the case of the liquid-liquid biphasic medium. The effect of water content was different according to the type of system used. The dependence of reaction rate and of conversion yield on enzyme and substrate concentrations was also investigated. From an applied point of view, the best performances were obtained with either microaqueous or liquid-liquid two-phase systems. The use of reverse micelles can be advocated only in particular conditions, such as low enzyme concentration, compatible with the specific constraints it involves.     

Over-stabilization of Candida antarctica lipase B by ionic liquids in ester synthesis

Four different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for butyl butyrate synthesis catalyzed by free Candida antarctica lipase B at 2% (v/v) water content and 50 °C. Lipase had enhanced synthetic activity in all ionic liquids in comparison with two organic solvents (hexane, and 1-butanol), the enhanced activity being related to the increase in polarity of ionic liquids. The continuous operation of lipase with all the assayed ionic liquids showed over-stabilization of the enzyme. The reuse of free lipase in 1-butyl-3-methylimidazolium hexafluorophosphate in continuous operation cycles showed a half-life time 2300 times greater than that observed when the enzyme was incubated in the absence of substrate (3.2 h), and a selectivity higher than 90%.     

Immobilization of lipase B from Candida antarctica on porous styrene-divinylbenzene beads improves butyl acetate synthesis

A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.     

Enzymatic esterification in organic media: role of water and organic solvent in kinetics and yield of butyl butyrate synthesis

Summary The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient. Offprint requests to: F. Monot     

Immobilization of Candida rugosa lipase in lyotropic liquid crystals and some properties of the immobilized enzyme

Summary Lipase from Candida rugosa has been immobilized in lyotropic liquid crystals consisting of a nonionic surfactant, hexane, and aqueous buffer with the enzyme. The kinetics of butyl butyrate synthesis, diffusion effects, and enzyme stability were investigated. Some basic rules have been formulated for a rational medium design in liquid-crystalline matrices.     

High yield lipase-catalyzed synthesis of Engkabang fat esters for the cosmetic industry

Engkabang fat esters were produced via alcoholysis reaction between Engkabang fat and oleyl alcohol, catalyzed by Lipozyme RM IM. The reaction was carried out in a 500 ml Stirred tank reactor using heptane and hexane as solvents. Response surface methodology (RSM) based on a four-factor-five-level Central composite design (CCD) was applied to evaluate the effects of synthesis parameters, namely temperature, substrate molar ratio (oleyl alcohol: Engkabang fat), enzyme amount and impeller speed. The optimum yields of 96.2% and 91.4% were obtained for heptane and hexane at the optimum temperature of 53.9 °C, impeller speeds of 309.5 and 309.0 rpm, enzyme amounts of 4.82 and 5.65 g and substrate molar ratios of 2.94 and 3.39:1, respectively. The actual yields obtained compared well with the predicted values of 100.0% and 91.5%, respectively. Meanwhile, the properties of the esters show that they are suitable to be used as ingredient for cosmetic applications.     

Optimization of Lipase-catalysed Synthesis of Butyl Butyrate Using a Factorial Design

Summary A lipase from Candida rugosa immobilized on styrene-divinylbenzene copolymer was used to catalyse the direct esterification of butanol and butyric acid. A factorial design was employed to evaluate the effects of temperature (37–50 °C), substrate molar ratio of butyric acid to butanol (0.6 to 2.0) and enzyme amount (0.2–0.4 g) on the ester yield. The main effects were fitted by multiple regression analysis to a linear model and maximum ester yield could be obtained working at 41 °C with 0.4 g of lipase. The mathematical model obtained, representing the ester yield has been found to describe adequately the experimental results. Under optimal conditions, concentration of 32.4 g butyl butyrate/l that corresponds to a yield of 75% was obtained.     

Enzymatic synthesis of butyl hydroxycinnamates and their inhibitory effects on LDL-oxidation

The potential of the Aspergillus niger type A feruloyl esterase (AnFaeA) for the synthesis of various phenolic acid esters was examined using a ternary-organic reaction system consisting of a mixture of n-hexane, 1- or 2-butanol and water. Reaction parameters including the type of methyl hydroxycinnamate, the composition of the reaction media, the temperature, and the substrate concentration were investigated to evaluate their effect on initial rate and conversion to butyl esters of sinapic acids. Optimisation of the reaction parameters lead to 78% and 9% yield for the synthesis of 1-butyl and 2-butyl sinapate, respectively. For the first time, a feruloyl esterase was introduced in the reaction system as cross-linked enzyme aggregates (CLEAs), after optimisation of the immobilisation procedure, allowing the recycling and reuse of the biocatalyst. The inhibition of copper-induced LDL oxidation by hydroxycinnamic acids and their corresponding butyl esters was investigated in vitro. Kinetic analysis of the antioxidation process demonstrates that sinapate derivatives are effective antioxidants indicating that esterification increases the free acid's antioxidant activity especially on dimethoxylated compounds such as sinapic acid compared to methoxy-hydroxy-compounds such as ferulic acid.     

Synthesisof fructose laurate esters catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst in a non-aqueous system

Earlier studies on fructose laurate ester products have shown that recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) on the cell surface acts as an efficient whole-cell biocatalyst for sugar ester production from fructose and lauric acid in an organic solvent. The effects of various reaction factors, including solvent composition, substrate molar ratio, enzyme dose, temperature and water activity, on esterification catalyzed by the CALB-displaying P. pastoris whole-cell biocatalyst were examined in the present study. Under the preferred reaction conditions, specifically, 5 mL organic solvent mixture of 2-methyl-2-butanol/DMSO (20% v/v), 2 mmol fructose with a lauric acid to fructose molar ratio of 2:1, 0.3 g whole-cell biocatalyst (1,264 U/g dry cell) with an initial water activity of 0.11, 1.2 g 4Å molecular sieve, reaction temperature of 55oC and 200 rpm stirring speed, the fructose mono laurate ester yield was 78% (w/w). The CALBdisplaying P. pastoris whole-cell biocatalyst exhibited good operational stability, with an evident increase, rather than decrease, in relative activity after the continuous recover and reuse cycle. The relative activity of the biocatalyst remained 50% higher than that of the first batch, even following reuse for 15 batches. Our results collectively indicate that the CALB-displaying P. pastoris whole-cell biocatalyst may be potentially utilized in lieu of free or immobilized enzyme to effectively produce non-ionic surfactants such as fatty acid sugar esters, offering the significant advantages of cost-effectiveness, good operational stability and mild reaction conditions.

     

Lipase-catalyzed acidolysis of fish liver oil with dihydroxyphenylacetic acid in organic solvent media

Lipase-catalyzed acidolysis reaction of fish liver oil with dihydroxyphenylacetic acid (DHPA) was investigated in terms of enzyme specificity as well as the effects of enzyme concentration, molar substrate ratio and organic solvent mixture on the bioconversion yield. The highest bioconversion yield of 83% was obtained when Novozym 435 was used as biocatalyst in a hexane:2-butanone mixture of 75:25 (v/v) at a fish liver oil to DHPA substrate molar ratio of 4:1; however, lower bioconversion yield (15%) was obtained when Lipozyme IM 20 was used. The bioconversion yield of phenolic monoacylglycerols (MAGs) increased from 11 to 70% when the ratio of the hexane/2-butanone reaction medium was changed from 85:15 to 75:25 (v/v), whereas that of phenolic diacylglycerols (DAGs) remained relatively unchanged (13–16%). The results also showed that the acidolysis reaction resulted in an increase of C_20:5 ω-3 and C_22:6 ω-3 proportions from 11.5 and 20.2% in the original fish liver oil to 22.6–27.1 and 22.8–23.1% in the phenolic lipids, respectively. The radical scavenging ability of phenolic lipids was determined to be about half-time lower than that of α-tocopherol.     

Study on response surface methodology (RSM) of lipase-catalyzed synthesis of palm-based wax ester

The synthesis of wax ester using refined, bleached and deodorized (RBD) palm oil and oleyl alcohol catalyzed by lipozyme IM was carried out. Response surface methodology (RSM) based on a five-level, four-variable central composite rotatable design (CCRD) was used to evaluate the interactive effects of synthesis, of reaction time (2.5–10 h), temperature (30–70 °C), amount of enzyme (0.1–0.2 g) and substrate molar ratio (palm oil to oleyl alcohol, 1:1–1:5) on the percentage yield of wax esters. The optimum conditions derived via RSM were: reaction time 7.38 h, temperature 53.9 °C, amount of enzyme 0.149 g, and substrate molar ratio 1:3.41. The actual experimental yield was 84.6% under optimum condition, which compared well to the maximum predicted value of 85.4%.     

Understanding structure-stability relationships of Candida antartica lipase B in ionic liquids

Two different water-immiscible ionic liquids (ILs), 1-ethyl-3-methylimidizolium bis(trifluoromethylsulfonyl)imide and butyltrimethylammonium bis(trifluoromethylsulfonyl)imide, were used for butyl butyrate synthesis from vinyl butyrate catalyzed by Candida antarctica lipase B (CALB) at 2% (v/v) water content and 50 degrees C. Both the synthetic activity and stability of the enzyme in these ILs were enhanced as compared to those in hexane. Circular dichroism and intrinsic fluorescence spectroscopic techniques have been used over a period of 4 days to determine structural changes in the enzyme associated with differences in its stability for each assayed medium. CALB showed a loss in residual activity higher than 75% after 4 days of incubation in both water and hexane media at 50 degrees C, being related to great changes in both alpha-helix and beta-strand secondary structures. The stabilization of CALB, which was observed in the two ILs studied, was associated with both the maintenance of the 50% of initial alpha-helix content and the enhancement of beta-strands. Furthermore, intrinsic fluorescence studies clearly showed how a classical enzyme unfolding was occurring with time in both water and hexane media. However, the structural changes associated with the incubation of the enzyme in both ILs might be attributed to a compact and active enzyme conformation, resulting in an enhancement of the stability in these nonaqueous environments.     

Short chain fatty acid esters synthesis by commercial lipases in low-water systems and by resting microbial cells in aqueous medium

Thirteen commercial lipases in hexane and seventeen bacterial cell suspensions in aqueous media were screened for the production of ethyl valerate and ethyl butyrate. The highest esterifying activity was obtained with commercial Pancrealipase (Biozymes Inc.) and Candida rugosa lipase (Amano Enzyme Ltd) and with bacterial cell suspension from Pseudomonas fragi CRDA 446. Commercial enzymes gave molar conversion yield of 68% over 24 h as compared to 17% with whole cells in aqueous medium. However, a comparison of both sources of biocatalyst i.e. whole microbial cells and commercial lipases, based on the amount of ester produced per g of protein for a complete reaction, indicated similar activities. © Rapid Science Ltd. 1998     

Effect of alcohol chain length on the optimum conditions for lipase-catalyzed synthesis of adipate esters

Immobilized Candida antarctica lipase B, Novozym® 435, was used in the esterification of adipic acid and alcohols with different chain lengths (C₁–C₁₈). Optimum conditions for the synthesis of adipate esters were obtained using response surface methodology (RSM) with respect to important reaction parameters including time, temperature, substrate molar ratio and amount of enzyme. Alcohol chain length specificity of the enzyme in the synthesis of adipate esters was also determined. Minimum reaction time (215 min) for achieving maximum ester yield was obtained for butyl alcohol. Methanol required an increased time (358 min) and enzyme amount (10.2%, w/w) for attaining maximum yield. The maximum required temperature and time of 65°C and 523 min, respectively, were obtained for the synthesis of dioctadecyl adipate. The results demonstrate that alcohol chain length is a determining parameter in optimization of the lipase-catalyzed synthesis of adipate esters. Reactions under optimized conditions yielded a high percentage of esterification (>97%). The optimum conditions can be used to scale up the process.     

Utilization of deep‐sea microbial esterase PHE21 to generate chiral sec‐butyl acetate through kinetic resolutions

We previously identified and characterized 1 novel deep‐sea microbial esterase PHE21 and used PHE21 as a green biocatalyst to generate chiral ethyl (S)‐3‐hydroxybutyrate, 1 key chiral chemical, with high enantiomeric excess and yield through kinetic resolution. Herein, we further explored the potential of esterase PHE21 in the enantioselective preparation of secondary butanol, which was hard to be resolved by lipases/esterases. Despite the fact that chiral secondary butanols and their ester derivatives were hard to prepare, esterase PHE21 was used as a green biocatalyst in the generation of (S)‐sec‐butyl acetate through hydrolytic reactions and the enantiomeric excess, and the conversion of (S)‐sec‐butyl acetate reached 98% and 52%, respectively, after process optimization. Esterase PHE21 was also used to generate (R)‐sec‐butyl acetate through asymmetric transesterification reactions, and the enantiomeric excess and conversion of (R)‐sec‐butyl acetate reached 64% and 43%, respectively, after process optimization. Deep‐sea microbial esterase PHE21 was characterized to be a useful biocatalyst in the kinetic resolution of secondary butanol and other valuable chiral secondary alcohols.     

Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.     

Optimized enzymatic synthesis of geranyl butyrate with lipase AY from Candida rugosa

Response surface methodology (RSM) and five-level, five-variable central composite rotatable design (CCRD) were used to evaluate the effects of synthetic variables, such as reaction time (1-9 h), temperature (25-65 degrees C), enzyme amount (10-50%), substrate molar ratio of geraniol to tributyrin (1:0.33-1:1), and added water amount (0-20%) on molar percent yield of geranyl butyrate, using lipase AY from Candida rugosa. Reaction time and temperature were the most important variables and substrate molar ratio had no effect on percent molar conversion. Based on contour plots, optimum conditions were: reaction time 9 h, temperature 35 degrees C, enzyme amount 50%, substrate molar ratio 1:0.33, and added water 10%. The predicted value was 100% and actual experimental value was 96.8% molar conversion. (c) 1996 John Wiley & Sons, Inc.     

Continuous lipase-catalyzed production of esters from crude high-oleic sunflower oil

The objective of this work was to develop an economically relevant enzymatic process of butyl ester production using crude high-oleic sunflower oil. Novozym 435, a non-regiospecific biocatalyst, provided the best compromise between activity and butyl-ester yield. The inhibition caused by the presence of phopholipids in crude oil was eliminated by using tert-butanol. It demonstrates the key role of the medium polarity in order to insure the stability of a process. Initial substrate concentrations and their molar ratio were optimized in a continuous packed-bed reactor to maximize product yield and productivity. The best compromise was obtained for an initial oil concentration of 500 mM and a molar ratio of 5. It enabled a high productivity of 13.8 tons year⁻¹ kg Novozym 435⁻¹ with a butyl-ester purity of 96.5% to be obtained. Experiments with the continuous reactor were performed over 50 days without any loss of enzyme activity.     

Enzymatic synthesis of benzyl benzoate using different acyl donors: Comparison of solvent-free reaction techniques

In this study, benzyl benzoate was successfully synthesized via enzymatic acylation using three immobilized enzymes as biocatalysts. Different acyl donors (benzoic acid and benzoic anhydride), operation regimes (batch, fed-batch), mixing modes (conventional mechanical stirring and ultrasound), process parameters (temperature, substrate molar ratio of acyl donor to acyl acceptor), presence or absence of solvents, enzyme amount and type were evaluated. Benzoic acid is a solid that is difficult to solubilize and, thus, was not efficient as acyl donor for the synthesis of benzyl benzoate. On the other hand, benzoic anhydride was very effective for the acylation of benzyl benzoate, and the presence of an excess of benzyl alcohol was essential to ensure the solute-solvent intermolecular attractions and good substrate solubilization, allowing the ester synthesis to be performed in the absence of organic solvents. The ultrasound was effective in increasing increase the initial reaction rate and the final conversion (88 %). However, the Lipozyme TL-IM and RM-IM supports were damaged, and the reuse was unfeasible. The batch and fed-batch approaches in conventional stirring ensured high conversions of 92 and 90 %, respectively, for batch (anhydride: alcohol 1:6) and fed-batch (1:3) using the Lipozyme TL-IM as biocatalyst. The controlled addition of the anhydride (fed-batch) allowed the reduction of alcohol molar ratio but decreased the reaction rates, and the maximum conversions were reached only after 24 h, while the batch approach had 92 % of conversion after 6 h. The yield of benzyl benzoate was high at 6 wt.% of enzyme, low temperature (50 °C), and simple reactor operation (batch). Results show the feasibility of the synthesis of benzyl benzoate via acylation using a green process that may be an alternative route to the chemical synthesis.     

Optimisation and Characterisation of Lipase-Catalysed Synthesis of a Kojic Monooleate Ester in a Solvent-Free System by Response Surface Methodology

Kojic acid is widely used to inhibit the browning effect of tyrosinase in cosmetic and food industries. In this work, synthesis of kojic monooleate ester (KMO) was carried out using lipase-catalysed esterification of kojic acid and oleic acid in a solvent-free system. Response Surface Methodology (RSM) based on central composite rotatable design (CCRD) was used to optimise the main important reaction variables, such as enzyme amount, reaction temperature, substrate molar ratio, and reaction time along with immobilised lipase from Candida Antarctica (Novozym 435) as a biocatalyst. The RSM data indicated that the reaction temperature was less significant in comparison to other factors for the production of a KMO ester. By using this statistical analysis, a quadratic model was developed in order to correlate the preparation variable to the response (reaction yield). The optimum conditions for the enzymatic synthesis of KMO were as follows: an enzyme amount of 2.0 wt%, reaction temperature of 83.69°C, substrate molar ratio of 1:2.37 (mmole kojic acid:oleic acid) and a reaction time of 300.0 min. Under these conditions, the actual yield percentage obtained was 42.09%, which is comparably well with the maximum predicted value of 44.46%. Under the optimal conditions, Novozym 435 could be reused for 5 cycles for KMO production percentage yield of at least 40%. The results demonstrated that statistical analysis using RSM can be used efficiently to optimise the production of a KMO ester. Moreover, the optimum conditions obtained can be applied to scale-up the process and minimise the cost.