Transesterification Rate Research Articles

Enzymatic transesterification of palm stearin: anhydrous milk fat mixtures using 1,3-specific and non-specific lipases

Abstract The physical characteristics of a palm stearin:anhydrous milkfat (40:60) mixture, enzymatically transesterified in a solvent-free system, was investigated by monitoring changes in the slip melting point (SMP), solid fat content (SFC) and melting characteristics. The enzymes used were 1,3-specific lipases from Aspergillus niger, Rhizomucor miehei, Rhizopus javanicus, Rhizopus niveus, Alcaligenes sp. and non-specific lipases from Pseudomonas sp. and Candida rugosa. Results indicated that Pseudomonas lipase-catalyzed mixtures produced the highest degree of transesterification (33.9%) and rate of transesterification (50.0/h), followed by R. miehei lipase at 32.3% and 27.1/h. The highest % free fatty acid (FFA) liberated was also from the reaction mixture catalyzed by Pseudomonas (2.6 1%) lipase followed by Alcaligenes (2.56%) and R. miehei (2.88%) lipases. The SMP of all the transesterified PS:AMF mixtures underwent only slight reductions, ranging from 0.5 to 2.5°C with reactions catalyzed by Pseudomonas and R. miehei lipases, producing the biggest drop in SMP values.

Site-specific DNA transesterification by vaccinia topoisomerase: role of specific phosphates and nucleosides.

Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-CCCTTp downward arrow in duplex DNA. Here we present experiments that illuminate the contributions of specific nucleosides and phosphates to site affinity and transesterification. We find that the -1 phosphate and -2 nucleoside on the scissile strand (5'-CCCTTp / NpN) enhance the rate of transesterification by factors of 40 and 25, respectively, whereas the DNA segment downstream of the -2 nucleotide makes no significant kinetic contribution. Placement of a 5'-phosphate/3'-OH nick at position +2, +3, +4, or +5 within the CCCTT element results in a 5-10-fold reduction in the affinity of topoisomerase binding to DNA. A nick at the +2 phosphate also slows the rate of transesterification by approximately 500-fold. This finding, together with earlier studies of the effects of position-specific base and sugar modifications, points to the +2 Tp nucleotide as being the most critical element of the CCCTT target site other than the scissile phosphate itself. On the noncleaved strand, the segment downstream of the 3'-GGGAA element contributes minimally to the rate of transesterification provided that the substrate is otherwise fully base-paired within the 5'-CCCTT target site. By studying the effects of single nucleotide gaps and missing phosphate nicks within the 3'-GGGAA sequence, we find that the +1 and +2 adenosine nucleosides enhance the rate of transesterification by 20- and 1,000-fold respectively and that the +5 phosphate (3'-GpGGAA) is also important for cleavage. Cumulative functional analyses of the vaccinia topoisomerase-DNA interface are discussed in light of newly available structures for the vaccinia and human type IB enzymes.

Transesterification in poly(ethylene terephthalate) and poly(ethylene naphthalene 2, 6-dicarboxylate) blends; the influence of hydroxyl end groups

Two samples of a single composition blend of PET and PEN were prepared by solution blending using different solvents such that the hydroxyl end groups in one blend were modified. The rate of transesterification in each blend was studied using proton NMR, an established technique for this system. The results indicate that the rate of transesterification is influenced dramatically by end group modification, providing clear evidence that hydroxyl end groups participate in the reaction mechanism.

Polyesters bearing furan moieties. Part 3. A kinetic study of the transesterification of 2-furoates as a model reaction for the corresponding polycondensations

The kinetics and mechanism of transesterification reactions involving furanic esters (and aliphatic or aromatic counterparts for the sake of comparison) have been studied in detail using various catalysts and a wide range of experimental conditions. The purpose of this model investigation is to gather a thorough understanding of the process which simulates the corresponding syntheses of furanic polyesters. Whereas the orders in the two reagents was consistently unity, the order with respect to the catalyst varied from 0.5 to about unity as a function of increasing temperature and of the structure of the promoter used. In the case of Ti(OBu)4 which was the catalyst most used, this behaviour was attributed to the existence of a monomer/dimer equilibrium of the catalyst molecules in the media selected for this work. The presence of the furan heterocycle produced a partial sequestration of the catalyst, through complexation induced by its dienic character, thus reducing the actual rate of transesterification. The existence of this complex was inferred by using unreactive furans as retarding additives and shown by FTIR spectroscopy. © 1999 Society of Chemical Industry

Kinetics of RNA Degradation by Specific Base Catalysis of Transesterification Involving the 2‘-Hydroxyl Group

A detailed understanding of the susceptibility of RNA phosphodiesters to specific base-catalyzed cleavage is necessary to approximate the stability of RNA under various conditions. In addition, quantifying the rate enhancements that can be produced exclusively by this common cleavage mechanism is needed to fully interpret the mechanisms employed by ribonucleases and by RNA-cleaving ribozymes. Chimeric DNA/RNA oligonucleotides were used to examine the rates of hydroxide-dependent degradation of RNA phosphodiesters under reaction conditions that simulate those of biological systems. Under neutral or alkaline pH conditions, the dominant pathway for RNA degradation is an internal phosphoester transfer reaction that is promoted by specific base catalysis. As expected, increasing the concentration of hydroxide ion, increasing the concentration of divalent magnesium, or raising the temperature accelerates strand scission. In most instances, the identities of the nucleotide bases that flank the target RNA linkage have a negligible effect on the pKa of the nucleophilic 2‘-hydroxyl group, and only have a minor effect on the maximum rate constant for the transesterification reaction. Under representative physiological conditions, specific base catalysis of RNA cleavage generates a maximum rate enhancement of ∼100 000-fold over the background rate of RNA transesterification. The kinetic parameters reported herein provide theoretical limits for the stability of RNA polymers and for the proficiency of RNA-cleaving enzymes and enzyme mimics that exclusively employ a mechanism of general base catalysis.

The Comparative Discriminating Abilities of Lipases in Different Media and Their Application in Fatty Acid Enrichment

The generally held belief that the selectivity of lipase can be changed by changing the media from aqueous to non-aqueous was tested by monitoring the rates of hydrolysis, ester synthesis and transesterification with a range of fatty acid mono-esters. Although the absolute rates of reaction varied, hydrolysis was by far the most rapid of the three, the relative rates for the fatty acids used were similar in all three reaction types. The selectivity of the five enzymes used appeared to remain unchanged irrespective of the type of reaction, i.e. hydrolysis of p-nitrophenyl esters, direct ester synthesis with butanol and fatty acid or transesterification with butyl butyrate and fatty acid, and could not be changed by changing water activity. This principle was applied to screen for suitable lipases which could be used to increase the gamma linolenic acid content of a fatty acid mixture. Enzymes could be selected by measuring the rate of hydrolysis of a range of P-nitrophenyl esters.

Effect of enzymatic transesterification on the fluidity of palm stearin-palm kernel olein mixtures

In an effort to improve the physical and/or melting characteristics of solid fats, the enzymatic transesterification of palm stearin-palm kernel olein (40:60) in a solvent-free system was investigated. The enzymes used were Celite-bound lipases that include 1,3-specific lipases such as Aspergillus niger, Alcaligenes sp. and nonspecific lipases such as Pseudomonas sp. and Candida rugosa. Commercial immobilized lipase from Rhizomucor miehei (Lipozyme 1M60) was also used. The efficacies of these enzymes for improving the melting behaviour of the oil mixtures were followed by slip melting point (SMP), solid fat content (SFC) and differential scanning calorimetry (DSC) analyses. Results indicated that enzymatic transesterification was able to produce fat mixtures with substantially lower melting points by repositioning the fatty acids of triglycerides in the higher melting range to form lower- or middle-melting components. Pseudomonas lipase-catalyzed mixtures produced the highest degree (152.2%) and rate (50.0h −1) of transesterification followed by R. miehei lipase at 151.7% and 27.1h −1, respectively. The highest % FFA liberated was also from the reaction mixture catalysed by Pseudomonas (2.90%) and R. miehei (2.54%) lipases. The Pseudomonas-catalyzed mixture also produced the biggest drop in SMP (12.0 °C) and the SFC results showed complete melting at 35 °C. Our findings also suggest that the positional specificity of lipases may not play a significant role in producing a more fluid product.

Effect of enzymatic transesterification on the melting points of palm stearin‐sunflower oil mixtures

AbstractTransesterification with lipases may be used to convert mixtures of fats to plastic fats, making them more suitable for use in edible products. In our study, 1,3‐specific (Aspergillus niger, Mucor javanicus, Rhizomucor miehei, Rhizopus javanicus, and Rhizopus niveus) and nonspecific (Pseudomonas sp. and Candida rugosa) lipases were used to transesterify mixtures of palm stearin and sunflower oil (PS‐SO) at a 40:60 ratio in a solvent‐free medium. The transesterified mixtures of PS‐SO were analyzed for their percentage free fatty acids (FFA), degree and rate of transesterification, solid fat content, slip melting point (SMP), and melting characteristics by differential scanning calorimetry. Results indicated that Pseudomonas sp. lipase produced the highest degree (77.3%) and rate (50.0 h−1) of transesterification, followed by R. miehei lipase at 32.7% and 27.1 h−1, respectively. The highest percentage FFA liberated was also in the reaction mixtures catalyzed by Pseudomonas sp. (2.5%) lipase and R. miehei (2.4%). Pseudomonas‐catalyzed mixtures produced the biggest drop in SMP (13.5°C) and showed complete melting at below body temperature. All results indicated conversion of the PS‐SO mixtures to a more fluid product. The findings also suggest that the specificity of lipases may not play a significant role in lowering the melting point of the PS‐SO mixtures.

Syntheses of Optically Active Polyacrylates

ABSTRACT Different secondary alcohols were resolved by lipase catalyzed transesterification using 2,3-butanedione monoxime acrylate as acrylating agent. The results showed that the reaction rates were the fastest among reactions reported until now. The effect of solvent on the transesterification rate was studied. The enantiomeric excess (ee) and enantiomeric purity (E value) of all the acrylate monomers were determined. The synthesized optically active acrylate monomers were polymerized by free radical polymerization technique.

Nonequal reactivity model for biocatalytic polytransesterification

AbstractThe lipase‐catalyzed polytransesterification of divinyl adipate and 1,4‐butanediol is a dynamic process controlled by the relative magnitudes of the rates of transesterification and hydrolysis as the polymerization proceeds. A mathematical model that describes the kinetics of the biocatalytic polytransesterification process is presented. Initial rate studies with model substrates in transesterification, hydrolysis, and esterification were used to provide the initial kinetic parameters that, when combined with the effect of enzyme specificity, were used to predict product compositions under different reaction conditions. A comparison of experimental data and model predictions shows that the product composition is strongly influenced by the selective nature of enzyme catalysis, and this model effectively predicts the results of a step condensation polymerization where reactivity depends on molecular weight.

Application of Novel Preparation Method for Surfactant-Protease Complexes Catalytically Active in Organic Media

We have applied the newly developed method of preparing surfactant-enzyme complexes to other different enzyme sources in order to study the efficacy of this technique in the catalyzed transesterification processes. Protease from varied material sources was modified with surfactant molecules utilizing water-in-oil (W/O) emulsions, and the complex so formed proved very effective in catalyzing vinyl butyrate transesterification with benzyl alcohol in organic media. By contrast, native commercial protease and a lyophilized protease from optimum buffer solution pH hardly catalyzed the above process. Although, conventionally prepared surfactant-coated protease had shown some catalytic activity, its performance was significantly lower than that of the surfactant-protease complex assembled in the novel preparation method. The typical transesterification rates catalytically induced by surfactant-protease complexes were in the range of 7-260-fold superior relative to other catalyzed reaction systems. The preparation and reaction conditions of the surfactant-protease P (Aspergillus melleus) have been optimized through studies of the effect of aqueous pH in the W/O emulsions, the nature of the organic solvents and surfactants, and the reaction temperature. The protease complex prepared from nonionic surfactant 2C 18 Δ 9 -GE at optimized aqueous pH yielded the best results in isooctane at 45 °C. This novel enzyme modification method is conceptually simple, and the resultant complexes are elucidable. It is hoped that this method will become useful, particularly in preparing enzymes that are nonaqueous media sensitive and soluble in organic solution.

Kinetic studies of fusarium solani pisi cutinase used in a gas/solid system: Transesterification and hydrolysis reactions

Fusarium solani cutinase supported onto Chromosorb P was used to catalyze transesterification (alcoholysis) and hydrolysis on short volatile alcohols and esters in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrates and removed reaction products simultaneously. A kinetic study was performed under differential operating conditions in order to get initial reaction rates. The effect of the hydration state of the biocatalyst on the kinetics was studied for 3 conditions of hydration (a(w) = 0.2, a(w) = 0.4 and a(w) = 0.6), the alcoholysis of propionic acid methyl ester with n-propanol, and for 5 hydration levels (from a(w) = 0.2 to a(w) = 0.6) for the hydrolysis of propionic acid methyl, ethyl or propyl esters. F. solani cutinase was found to have an unusual kinetic behavior. A sigmoid relationship between the rate of transesterification and the activity of methyl propionate was observed, suggesting some form of cooperative activation of the enzyme by one of its substrate. For the hydrolysis of short volatile propionic acid alkyl esters, threshold effects on the reaction rate, highly depending on the water activity and the substrate polarity, are reported.

Kinetic studies of fusarium solani pisi cutinase used in a gas/solid system: Transesterification and hydrolysis reactions

Fusarium solani cutinase supported onto Chromosorb P was used to catalyze transesterification (alcoholysis) and hydrolysis on short volatile alcohols and esters in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrates and removed reaction products simultaneously. A kinetic study was performed under differential operating conditions in order to get initial reaction rates. The effect of the hydration state of the biocatalyst on the kinetics was studied for 3 conditions of hydration (aw = 0.2, aw = 0.4 and aw = 0.6), the alcoholysis of propionic acid methyl ester with n-propanol, and for 5 hydration levels (from aw = 0.2 to aw = 0.6) for the hydrolysis of propionic acid methyl, ethyl or propyl esters. F. solani cutinase was found to have an unusual kinetic behavior. A sigmoid relationship between the rate of transesterification and the activity of methyl propionate was observed, suggesting some form of cooperative activation of the enzyme by one of its substrate. For the hydrolysis of short volatile propionic acid alkyl esters, threshold effects on the reaction rate, highly depending on the water activity and the substrate polarity, are reported. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 1–8, 1997.

Degenerate Transesterification of Dimeric Lithium 2,4,6-Trimethylphenolate and a Further Observation on the Reaction of Tetramers1

The rates of exchange of the 2,4,6-trimethylphenolate ion between dimeric lithium 2,4,6-trimethylphenolate-d9 and a series of 2,4,6-trimethylphenyl esters [2,4,6-(CH3)3C6H2COOCH2R; R = CH2CH3, CH3O, Ph, 4-CH3Ph, 4-CF3Ph, 4-ClPh, 4-CH3OPh, 2-pyridyl, and 4-pyridyl] have been determined in dioxolane, THF, 1,2-dimethoxyethane, and pyridine. The reactions are first order in the lithium phenolate showing that the dimer reacts without dissociation. The rates of transesterification increase with increasing solvent donicity in contrast to the reaction of tetrameric lithium 3,5-dimethylphenolate. For all the esters, except R = 2-pyridyl, the rates of transesterification exhibit a linear correlation with their rates of hydrolysis. The 2-pyridyl ester exhibits an abnormally high rate of transesterification, which is attributed to a complex-induced proximity effect. Tetrameric lithium 3,5-dimethylphenolate undergoes transesterification less rapidly in the presence of hexamethylphosphoric triamide (0.25−1.6 equiv), a ...

Mutational analysis of vaccinia virus topoisomerase identifies residues involved in DNA binding.

Vaccinia DNA topoisomerase catalyzes the cleavage and re-joining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate formed at a specific target sequence, 5'-(C/T)CCTT downward arrow. The 314 aa protein consists of three protease-resistant structural domains demarcated by protease-sensitive interdomain segments referred to as the bridge and the hinge. The bridge is defined by trypsin-accessible sites at Arg80, Lys83 and Arg84. Photocrosslinking and proteolytic footprinting experiments suggest that residues near the interdomain bridge interact with DNA. To assess the contributions of specific amino acids to DNA binding and transesterification chemistry, we introduced alanine substitutions at 16 positions within a 24 aa segment from residues 63 to 86(DSKGRRQYFYGKMHVQNRNAKRDR). Assays of the rates of DNA relaxation under conditions optimal for the wild-type topoisomerase revealed significant mutational effects at six positions; Arg67, Tyr70, Tyr72, Arg80, Arg84 and Asp85. The mutated proteins displayed normal or near-normal rates of single-turnover transesterification to DNA. The effects of amino acid substitutions on DNA binding were evinced by inhibition of covalent adduct formation in the presence of salt and magnesium. The mutant enzymes also displayed diminished affinity for a subset of cleavage sites in pUC19 DNA. Tyr70 and Tyr72 were subjected to further analysis by replacement with Phe, His, Gln and Arg. At both positions, the aromatic moiety was important for DNA binding.

Functionalized dendritic polybenzylethers as acid/base buffers for biocatalysis in nonpolar solvents

A carboxylic acid functionalized dendritic polybenzyl ether has been synthesized and used with its sodium salt to generate a novel acid/base buffer soluble in nonpolar organic solvents. The effect of different ratios of the two buffer forms on the catalytic activity of subtilisin Carlsberg and chymotrypsin was investigated in toluene. It was found that reproducible transesterification rates were obtained at each molar ratio consistent with a buffering effect. As the molar ratio of the sodium salt to acid was increased there was a corresponding increase in the catalytic activity of both enzymes although their profiles were not identical. This is consistent with a requirement for deprotonation of a residue at active site of the enzyme as observed in aqueous solution. The ability to alter and precisely control the ionization state of biocatalysts in nonpolar solvents may find useful applications for both fundamental studies and in syntheses where reactants or products have acid/base properties. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 278-282, 1997.

The effect of block structure of an ethylene terephthalate/hydroxybenzoate copolymer on its ability to form compatible blends

AbstractA thermotropic liquid crystalline polymer (TLCP) containing aliphatic linkages exhibits properties of both a random‐coil and a rigid‐rod. By varying its block structure, one can change its ability to form compatible blends with other polymers. In this work, the ability of the TLCP p(ET/HBA60) with a high degree of block structure, X‐7GTM, to form compatible blends with polycarbonate and aromatic TLCPs is compared to that of a p(ET/HBA60) with a more random molecular structure, RodrunTM LC‐3000. It was found that both materials formed compatible blends through transesterification of the block structure and that a higher degree of blockiness enhanced the rate of transesterification. NMR spectra and differential thermal analysis were used to quantify the degree of transesterification and scanning electron microscopy was utilized to examine the effects of the blending process on the degree of compatibilization and the morphology of the resulting blends.

Synthesis of alkyds involving regioselective lipase-catalyzed transesterification in organic media

Porcine pancreatic lipase (PPL) is established as an effective biocatalyst for the selective transesterification of triglycerides and diols in organic medium at room temperature yielding primarily 2-monoglyceride mixtures. Molecular modelling simulations of repeating units of alkyd resin incorporating one- and two-monoglycerides show end-to-end distance of 9.76 and 12.82 A, respectively. The modeling studies on the trimers of these repeating units also reveal more extended configuration for the structures based on two-monoglycerides. A comparative study of alcoholysis by conventional base-catalyzed process and this novel biocatalytic process was carried out with coconut oil triglycerides and a series of diols, followed by polymerization. The rates of transesterification were dependent on the nature of triglyceride, diol, concentration of the enzyme, and temperature. The 1,3 specificity of lipase produced 2-monoglycerides as determined by periodic acid method, resulting in uniform distribution of fatty acids along the alkyd polyester backbone. Comparisons are made between those alkyds prepared by base catalysis and biocatalytic method. © 1997 John Wiley & Sons, Inc.

Effects of Mixed Anionic and Cationic Surfactants on Rate of Transesterification and Hydrolysis of Esters

The effects of mixed sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTABr) surfactants on pseudo first-order rate constants (kobs) for methanolysis of ionized phenyl salicylate (PS−) and alkaline hydrolysis of phenyl benzoate (PB) reveal the presence of micelle type aggregates or micelles, anionic in nature whenX≥ 4 and cationic in nature whenX≤ 0.3, whereX= [SDS]/[CTABr].

Interfacial structure in reacting poly(methyl methacrylate)/polycarbonate polymer blends

AbstractThe snall angle neutron scattering (SANS) technique has been used to study the interfacial structure development in poly(methyl methacrylate)/polycarbonate (PMMA/PC) blends through transesterification. We found: (1) Development of interfacial structure through transestrification at the phase separated domain interfaces are observed at T ≤ 200°C. At T ≥ 215°C, because of a higher transesterification rate, the melting of domains is observed together with very weak scattering by interfacial structure. (2) Interfacial thickness ti increases with time and reaches a plateau value of ti at 32.5 ± 2.4 Å and 29.6 ± 3.7 Å for 180°C and 200°C, respectively. (3) The internsity profiles at 190°C and long times shows the formation of spherical domains similar to those reported by Rabeony et al. (1). These may be caused by the crosslinking effect of the transesterification reaction between PMMA and PC.