Chemometric analysis of lipase-catalyzed synthesis of xylitol esters in a solvent-free system

Immobilized Candida antarctica lipase B-catalyzed esterification of xylitol and two fatty acids (capric and caproic acid) were studied in a solvent-free system. The Taguchi orthogonal array method based on three-level-four-variables with nine experiments was applied for the analysis and optimization of the reaction parameters including time, substrate molar ratio, amount of enzyme, and amount of molecular sieve. The obtained conversion was higher in the esterification of xylitol and capric acid with longer chain length. The optimum conditions derived via the Taguchi approach for the synthesis of xylitol caprate and xylitol caproate were reaction time, 29 and 18 h; substrate molar ratio, 0.3 and 1.0; enzyme amount, 0.20 and 0.05 g, and molecular sieve amount of 0.03 g, respectively. The good correlation between the predicted conversions (74.18% and 61.23%) and the actual values (74.05% and 60.5%) shows that the model derived from the Taguchi orthogonal array can be used for optimization and better understanding of the effect of reaction parameters on the enzymatic synthesis of xylitol esters in a solvent-free system.     

Optimized lipase-catalyzed synthesis of adipate ester in a solvent-free system

Immobilized Candida antarctica lipase-catalyzed esterification of adipic acid and oleyl alcohol was investigated in a solvent-free system (SFS). Optimum conditions for adipate ester synthesis in a stirred-tank reactor were determined by the response surface methodology (RSM) approach with respect to important reaction parameters including time, temperature, agitation speed, and amount of enzyme. A high conversion yield was achieved using low enzyme amounts of 2.5% w/w at 60°C, reaction time of 438 min, and agitation speed of 500 rpm. The good correlation between predicted value (96.0%) and actual value (95.5%) implies that the model derived from RSM allows better understanding of the effect of important reaction parameters on the lipase-catalyzed synthesis of adipate ester in an organic solvent-free system. Higher volumetric productivity compared to a solvent-based system was also offered by SFS. The results demonstrate that the solvent-free system is efficient for enzymatic synthesis of adipate ester.     

Lipase-catalyzed synthesis of chlorogenate fatty esters in solvent-free medium

Chlorogenic acid (5-caffeoylquinic acid or 5-CQA) is an hydrophilic phenolic compound with antioxidant properties. Because of its high polarity, its antioxidant properties may be altered when formulated in oil based food or cosmetic preparations. Therefore, there is an interest in trying to enhance its hydrophobicity by grafting of an aliphatic chain. Such lipophilization reactions can be generally achieved through enzymatic catalysis. Our study consisted in synthesizing fatty cholorogenate esters in a two steps reaction. Firstly, 5-CQA was chemically esterified by methanol using an Amberlite IR120 H resin to obtain methyl chlorogenate that is more soluble in the fatty alcohols than 5-CQA. Secondly, this chlorogenate intermediate was transesterified with fatty alcohols of various chain lengths (C₄, C₈, C₁₂, or C₁₆) in the presence of Candida antarctica B lipase. Under optimal reaction conditions (a_w = 0.05; 5% (w/w) of biocatalyst), the transesterification rates were until two-fold higher than in the direct lipase-catalyzed esterification of chlorogenic acid by the same alcohols. The two-step reaction overall yield was between 61 and 93% depending on the alcohol chain length, whereas it was 40–60% for the direct esterification with the same alcohols.     

Enzymatic synthesis of trieicosapentaenoylglycerol in a solvent-free medium

The enzymatic synthesis of trieicosapentaenoylglycerol from glycerol and eicosapentaenoic ethyl ester in a solvent-free medium is studied here. Novozym SP 435 (immobilized lipase from Candida antarctica) has appeared as a very efficient biocatalyst for this transesterification. A nitrogen bubbling has allowed a good mixing and also the shifting of the reaction toward synthesis by eliminating the ethanol formed. The effect of temperature and of the quantity of lipase has been studied. In the optimal conditions (T=80°C, 5% (w/w) of lipase, 1 mol glycerol for 3 mol ethyl ester), pure triglyceride has been obtained after 10 h.     

Parameters affecting incorporation and by-product formation during the production of structured phospholipids by lipase-catalyzed acidolysis in solvent-free system

By-product formation is a serious problem in the lipase-catalyzed acyl exchange of phospholipids (PL). By-products are formed due to parallel hydrolysis reactions and acyl migration in the reaction system. A clear elucidation of these side reactions is important for practical operation in order to minimize by-products during reaction. In the present study we examined the lipozyme RM IM-catalyzed acidolysis for the production of structured phospholipids between phosphatidylcholine (PC) and caprylic acid in the solvent-free system. A five-factor response surface design was used to evaluate the influence of major factors and their relationships on a number of responses reflecting the turnover of main reactions as well as side reactions. The five factors, including enzyme dosage, reaction time, reaction temperature, substrate ratio (mol/mol caprylic acid/PC) and water addition, were varied at three levels with two star points. All parameters besides water addition had an effect on the incorporation of caprylic acid into PC and lysophosphatidylcholine (LPC). Reaction time and enzyme dosage showed increased effect on incorporation into PC, while substrate ratio and reaction temperature showed opposite effect. The PC content decreased with increase of all parameters except for substrate ratio. Optimal conditions are recommended as enzyme dosage 40%, reaction temperature 55 °C, water addition 1%, reaction time 70 h, and substrate ratio 6 mol/mol caprylic acid/PC. Under these conditions an incorporation of 46% with PC accounting for 53% of the PL fraction can be obtained. Regiospecific analysis of the product revealed that the caprylic acid was mainly incorporated into the sn-1 position accounting for 80% of the fatty acids incorporated.     

Lipase catalysed synthesis of sebacic and phthalic esters

The enzymatic synthesis and hydrolysis of alkyl sebacates and o-, m-, p-phthalates were studied. Biosyntheses were conducted through alcoholysis of dimethyl phthalates and dimethyl sebacate with 2-ethylhexanol and 3,5,5-trimethylhexanol in a solvent-free medium, using lipases from Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme IM) and Porcine pancreas (PPL). It was found that the synthesis and hydrolysis of sebacic acid esters were characterised by a satisfactory rate, however, by low enantioselectivity. The yield of synthesis of di-3,5,5-trimethylhexyl sebacate catalysed by Novozym 435 at 50 °C was 84%, after 20 h of reaction. The degree of conversion, 62.9% after 350 h, was obtained for alcoholysis reaction of dimethyl m-phthalate with 3,5,5-trimethylhexanol. For the enzymes used, no activity was detected at all on both the synthesis and hydrolysis of di-2-ethylhexyl o-phthalate and di-3,5,5-trimethylhexyl o-phthalate.     

Kinetics of the lipase-catalyzed synthesis of glucose esters in acetone

A simple kinetic model derived from a ping-pong bi-bi mechanism is proposed to describe the lipase-catalyzed esterification of glucose with fatty acids. The mathematical expressions derived from this model have been tested using several sets of data obtained from reactions carried out under different reaction conditions. The predicted values provide very good fits of the experimental data for temperatures from 30 to 60 degrees C, enzyme loadings from 90 to 180 mg, and fatty acid concentrations from 0.33M to 1M. Experiments conducted at different temperatures permit one to estimate an activation energy of approximately 12 kcal/mol for the rate-limiting step of the reaction (formation of the acyl-enzyme complex). The model also considers the kinetics of inactivation of the biocatalyst during the reaction.     

Enzymatic synthesis of isoamyl butyrate using immobilized Rhizomucor miehei lipase in non-aqueous media

Isoamyl butyrate, an important fruity flavor ester, was synthesized using Rhizomucor miehei lipase immobilized on a weak anion exchange resin (Lipozyme IM-20) by the esterification of isoamyl alcohol and butyric acid. The effects of various reaction parameters such as substrate and enzyme concentrations, substrate molar ratio, temperature and incubation time have been investigated. Yields above 90% were obtained with substrate concentrations as high as 2.0 M. No evidence of enzyme inhibition by butyric acid was present up to 1.0 M concentration. Acid inhibition and, to a small extent, alcohol inhibition were evident above 1.0 M substrate concentration. Conversions reached a saturation value by the end of 24–48 h of incubation due to the accumulation of the water of reaction. The equilibrium was successfully pushed forward towards esterification by removing the accumulated water using a molecular sieve.Journal of Industrial Microbiology & Biotechnology (2000) 25, 147–154. Received 09 February 2000/ Accepted in revised form 24 June 2000     

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.     

Lipase catalysed synthesis of aliphatic, terpene and aromatic esters by alcoholysis in solvent-free medium

Biosynthesis of pentyl and terpene valerates, terpene acetates, hexyl and benzyl benzoates was conducted via alcoholysis, at 55 °C, in a solvent-free medium, using lipases from Candida antarcticaand Mucor mieheias catalysts. The yield higher than 90% was achieved (Candida antarctica)after 10 h for synthesis of pentyl valerate and 3-methylbutyl valerate, 35 h for 2-methylbytyl valerate, 6 h for geranyl and cytronelyl acetates, 10 h for geranyl and cytronelyl valerates, 75 h for hexyl benzoate and 100 h for benzyl benzoate.     

Enzymatic production of enantiopure ketoprofen in a solvent-free two-phase system

Enzymatic hydrolysis conducted in a medium composed of solely substrate is considered to resolve racemic ketoprofen esters. In a system composed of two components, the pure liquid substrate (organic phase) and water (aqueous phase), hydrolysis products can be efficiently removed from the reaction mixtures. Accordingly, in this study we designed a solvent-free two-phase system for the enantioselective enzymatic hydrolysis of ketoprofen esters. In order to further optimize this system, the influences of various factors, such as the pH of the aqueous phase, temperature, enzyme content, and the alcohol chain length of esters, were examined on conversion and enantiomeric excess. 1N NaHCO₃ was identified as the most efficient aqueous phase for the extraction of ketoprofen. Changes in the amount of enzyme did not significantly affect the maximum conversion or the enantiomeric excess. On the other hand, ketoprofen esters with shorter alcohol chains displayed higher initial reaction rates and conversions in solventless media. In the case of ketoprofen propyl ester, for example, the productivity of the solvent-free two-phase system was about 10–100 times higher than that obtained to date for ketoprofen esterification with alcohols in organic solvents. The enantioselectivities obtained in solvent-free media were similar to those obtained for the enantioselective esterification of ketoprofen in organic solvents.     

Enzymatic synthesis of the acrylic esters: a comparative study

Various acrylic esters were synthesized by Candida cylindracea lipase (CCL) catalysed transesterification with different alcohols. A comparative study was carried out using 2,3-butanedione mono-oxime acrylate and vinyl acrylate as acylating agents. The rate of conversion was faster when oxime acrylate was used as acylating agent as compared to vinyl acrylate. Effect of solvents on the rate of conversion was studied and diisopropyl ether (DIPE) was proved to be a better solvent as compared to CHCl₃ and THF. The effect of various structural aspects of the substrates on the rate of conversion was studied. Among the linear alcohols studied, ease of conversion was found to be in the order of n-octanol>n-hexanol>n-butanol. Up to 80% conversion was achieved in the case of cyclohexyl methanol.     

Recent developments in lipase-catalyzed synthesis of polymeric materials

In the past three years, enzymatic polymerization has dramatically developed and provided many successful examples in the construction of functional polymeric materials. In this review, the lipase-catalyzed synthesis of polymeric materials is systematically summarized, focusing on the synthesis of complex and well-defined polyesters. Exploration of novel biocatalysts and reaction media is described, with particular emphasis on the enzymes obtained via immobilization or protein engineering strategies, green solvents and reactors. Enzymatic polyester synthesis is then discussed with regard to the different reaction types, including ring-opening polymerization, polycondensation, combination of ring-opening polymerization with polycondensation, and chemoenzymatic polymerization. Using enzymatic polymerization, many polymeric materials with tailor-made structures and properties have been successfully designed and synthesized. Finally, recent developments in catalytic kinetics and mechanistic studies through the use of spectroscopy, mathematics and computer techniques are introduced. Overall, the review demonstrates that lipase-catalyzed synthesis of polymeric materials could be a promising platform for green polymer chemistry, and will be potential to produce biodegradable and biocompatible polymers.     

Adverse effect of chloride impurities on lipase-catalyzed transesterifications in ionic liquids

The adverse influence of chloride impurities on the lipase-catalyzed transesterification in ionic liquid is described. The activity of lipase from Rhizomucor miehei exponentially decreased with increasing Cl⁻ content in 1-octyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl] amide, [Omim][Tf₂N], and the activity of lipase in [Omim][Tf₂N] mixture containing 2% [Omim] [Cl] was only about 2% of the activity in pure [Omim][Tf₂N]. The activity of lipase from Candidantarctica linearly decreased at about 5% with every 1% increase in [Omim][Cl] with there being no activity in [Omim][Tf₂N] containing about 20% [Omim][Cl].     

Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: II. Effect of temperature on reaction kinetics and general considerations of stability and productivity

Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RrnL), isolated from commercial preparations of Lipolase and Lipozyme, respectively, were solubilized in AOT-stabilized water-in-oil (w/o) microemulsions in n-heptane and aspects of their hydrolysis and condensation activity examined. The temperature dependence of HIL hydrolysis activity in unbuffered R = 10 microemulsions matched very closely that for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. Apparent activation energies were measured as 13 +/- 2 and 15 +/- 2 kJ mol / respectively. Condensation activity, however, was essentially independent of temperature over the range 5 degrees to 37 degrees C. The stability of HIL over a 30-day period was very good at all pH levels (6.1, 7.2, 9.3) and R values studied (5, 7.5, 10, 20), except when high pHs and low R values were combined. The excellent stability was reflected by the linearity of the productivity profiles which facilitate system optimization. The temperature dependence of RmL hydrolysis activity toward pNPC(4) showed a maximum at 40 degrees C and an apparent E(act) = 20 +/- 2 kJ mol(-1) was calculated based on the linear region of the profile (5 degrees to 40 degrees C). RmL esterification activity showed only a slight dependence on temperature over the studied range (0 degrees to 40 degrees C) and an apparent E(act) = 5 +/- 1 kJ mol(-1) was measured for octyl decanoate synthesis. Both RmL and HIL, therefore, have potential for application in low temperature biotransformations in microemulsion-based media. The stability of RmL over a 30-day period was good in R = 7.5 and R = 10 microemulsions containing pH 6.1 buffer, and this was reflected in the linearity of their respective productivity profiles. RmL stability was markedly poorer at more alkaline pH, however, and proved to be sensitive to relatively small changes in the R value. (c) 1995 John Wiley & Sons, Inc.     

Enzymic peptide synthesis in microaqueous, solvent-free systems

Thermolysin-catalyzed (EC 3.4.24.4) and chymotrypsin-catalyzed (EC 3.4.21.1) peptide synthesis reactions were accomplished without any organic solvent in the presence of low amounts of water under sonication and fluidization. The systems used are considered to be microaqueous solvent-free ones. The influence of several reaction parameters, such as time, the amount of enzyme, the amount of water in free form or bound as hydration water, and the N/C component ratio, on the vield of the thermolysin-catalyzed synthesis of Z-Phe-Leu-NH(2) (up to 87% yield) was investigated in a sonicated system. Besides Z-Phe-Leu-NH(2), the tripeptide derivatives Ac-Xaa-Trp-Leu-NH(2), (Xaa = Gly, Ala) were also obtained in good yields of 79 and 71% respectively. In the latter case, no hydrolytic side reactions were observed. Using a fluidized-bed reactor, chymotrypsin- and thermolysin-catalyzed syntheses of N-protected di- and tripeptide amides could be perfromed with yields in the range of 10 to 40%. (c) 1995 John Wiley & Sons, Inc.     

Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: I. Effect of pH and water content on reaction kinetics

Lipolase and Lipozyme are produced in large quantities (as a result of genetic engineering and overexpression) for the detergents market and provide a cheap source of highly active biocatalysts. Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RmL) have been isolated in partially purified form from commercial preparations of Lipolase and Lipozyme, respectively. These lipases were solubilized in Aerosol-OT (AOT)-stabilized water-in-oil (w/o) microemulsions in n-heptane. HIL and RmL activity in these microemulsions was assayed by spectrophotometric measurement of the initial rate of p-nitophenyl butyrate hydrolysis, and by chromatographic determination of the initial rate of octyl decanoate synthesis from 1-octanol and decanoic acid. The hydrolytic activity of HIL in microemulsions measured as a function of buffer pH prior to dispersal, followed a sigmoidal profile with the highest activities observed at alkaline pHs. This broadly matches the pH-activity profile for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. The hydrolytic activity of RmL in the same microemulsions, measured as a function of pH, gave a bell-shaped profile with a maximum activity at pH 7.5. Again, the observed pH-activity profile was similar to that reported for a purified RmL in a tributyrin-based aqueous emulsion assay. In contrast, the esterification activity exhibited by both HIL and RmL in AOT microemulsions over the available range pH 6.1 to 10.4, decreases as the pH increases, most likely reflecting the effect of substrate ionization. The dependence of the hydrolytic and condensation activity of HIL on R, the mole ratio of water to surfactant, were similar with both profiles exhibiting a maximum at R = 5. The hydrolytic and esterification activities of RmL followed similar R-dependent profiles, but the profiles in this case exhibited a maximum at R = 10. The water activities at these R values were directly measured as 0.78 and 0.9, respectively. Measured water activities were unperturbed by the presence of lipase at the concentrations used in these studies. (c) 1995 John Wiley & Sons, Inc.     

Continuous lipase-catalyzed production of fatty acid ethyl esters from soybean oil in compressed fluids

This work investigates the continuous production of fatty acid ethyl esters from soybean oil in compressed fluids, namely carbon dioxide, propane and n-butane, using immobilized Novozym 435 as catalyst. The experiments were performed in a packed-bed bioreactor evaluating the effects of temperature in the range of 30–70 °C, from 50 to 150 bar, oil to ethanol molar ratio of 1:6–1:18 and solvent to substrates mass ratio of 4:1–10:1. In contrast to the use of carbon dioxide and n-butane, results showed that lipase-catalyzed alcoholysis in a continuous tubular reactor in compressed propane might be a potential route to biodiesel production as high reaction conversions were achieved at mild temperature (70 °C) and pressure (60 bar) conditions in short reaction times.     

Preparation of astaxanthin by lipase-catalyzed hydrolysis from its esters in a slug-flow microchannel reactor

Natural astaxanthin is widely used as a food and cosmetics additive because of its multiple biological activities. However, astaxanthin produced by Haematococcus pluvialis is generally esterified, and its activity is far less than that of free astaxanthin. Hydrolysis of astaxanthin esters to free astaxanthin by enzymes can overcome the drawbacks of chemical saponification methods. In this paper, a slug-flow microchannel reactor was constructed and tested in enzymatic hydrolysis of astaxanthin esters. The reactor consists of a “T” slug-flow generator, a stainless-steel microchannel, two constant-flow pumps, and a temperature controller. The reactor has the advantages of simple configuration and easy scale-up, and is suitable for two-phase biochemical reactions. Using the microchannel reactor, astaxanthin esters in H. pluvialis oil were efficiently hydrolyzed to free astaxanthin by lipase from Aspergillus niger. After hydrolysis, the content of free astaxanthin in H. pluvialis oil was 18.8 mg/L, 7.83-times higher than that before hydrolysis (2.13 mg/L). The hydrolysis rate reached 75.4 %. These results indicate that the microchannel reactor can be useful for the production of free astaxanthin from its esters.     

Amino acid pyridoxyl esters in peptide synthesis

Pyridoxyl residue was suggested to be used as a multifunctional protective and modifying group in peptide synthesis. The modification was carried out by introducing the pyridoxyl residue in free or partially protected peptides or by the addition of amino acid pyridoxyl esters by the methods of conventional peptide synthesis without the removal of the pyridoxyl group at the terminal stages of the synthesis (the second approach is more convenient). Pyridoxyl residue was also used as a spacer in solid phase peptide synthesis. It was attached to the polymer by the alkylation of the hydroxyl groups or of the pyridine ring of the pyridoxyl derivatives with the chloromethylated styrene-divinylbenzene copolymer (the standard Merrifield resin). Potentials for the use of pyridoxyl derivatives in the synthesis of linear, multiplet, and cyclic peptides are discussed.