Lipase/esterase-catalyzed ring-opening polymerization: A green polyester synthesis technique

In the last decade, there has been increased interest in lipase/esterase-catalyzed ring-opening polymerization as an alternative to metal-based catalytic processes. This review focuses on three components in the reaction system, namely biocatalysts, reaction medium and monomers. Novel lipases or esterases are described with particular emphasis on, those derived from thermophiles, immobilized enzymes and recombinant whole-cell biocatalysts. Green solvents in enzymatic ring-opening polymerization, including water, ionic liquids, supercritical carbon dioxide and hydrofluorocarbon solvents, are also discussed. Enzymatic ring-opening polymerization is reviewed with regard to the variety of polymers obtainable, such as polyesters, polycarbonates, polyphosphates and polythioesters. Among these, enzymatic synthesis of polyesters has been most widely investigated, and is discussed for lactones with small to large ring sizes. Finally, the mechanism of enzymatic ring-opening polymerization is described, which is generally accepted as a monomer-activated mechanism. Overall, the review demonstrates that lipase/esterase-catalyzed synthesis of polymers via ring-opening polymerization provides an effective platform for conducting “green polymer chemistry”.     

Modeling of lipase catalyzed ring-opening polymerization of epsilon-caprolactone

Enzymatic ring-opening polymerization of epsilon-caprolactone by various lipases was investigated in toluene at various temperatures. The determination of molecular weight and structural identification was carried out with gel permeation chromatography and proton NMR, respectively. Among the various lipases employed, an immobilized lipase from Candida antartica B (Novozym 435) showed the highest catalytic activity. The polymerization of epsilon-caprolactone by Novozym 435 showed an optimal temperature of 65 degrees C and an optimum toluene content of 50/50 v/v of toluene and epsilon-caprolactone. As lipases can degrade polyesters, a maximum in the molecular weight with time was obtained due to the competition of ring opening polymerization and degradation by specific chain end scission. The optimum temperature, toluene content, and the variation of molecular weight with time are consistent with earlier observations. A comprehensive model based on continuous distribution kinetics was developed to model these phenomena. The model accounts for simultaneous polymerization, degradation and enzyme deactivation and provides a technique to determine the rate coefficients for these processes. The dependence of these rate coefficients with temperature and monomer concentration is also discussed.     

Lipase catalyzed synthesis of cinnamyl acetate via transesterification in non-aqueous medium

Cinnamyl acetate is used as flavor and fragrance ingredient in food and cosmetic industries. This work focuses on the synthesis of cinnamyl acetate via lipase catalyzed transesterification of cinnamyl alcohol with vinyl acetate in non-aqueous medium. Among the different immobilized lipases employed, Novozym 435 was found to be the best catalyst in toluene as solvent. The effects of various parameters were studied systematically. With a mole ratio of 1:2 of cinnamyl alcohol to vinyl acetate and 10 mg catalyst, 96% conversion was obtained in 1 h at 40 °C. The ternary complex mechanism with inhibition by cinnamyl alcohol was found to fit the data well. The kinetics of the reaction was studied by using non-linear regression analysis. Enzymatic synthesis of cinnamyl acetate is an efficient process vis-à-vis chemical catalysis.     

Lipase catalyzed formation of flavour esters

Summary Thirteen commercial lipase preparations were checked for their ability to catalyse the formation of flavour esters (isoamyl or geranyl acetate, propionate and butyrate) by either direct esterification or ester solvolysis in n-heptane. The formation of isoamyl or geranyl butyrates and propionates by direct esterification was catalyzed by the majority of the tested lipases. Acetic acid esters were more difficult to obtain. Transesterification reactions were found to be a good alternative way for ester synthesis.     

Lipase catalyzed transesterification of soybean oil using ethyl acetate,an alternative acyl acceptor

Methanol, the acyl acceptor usually used in the commercial process of biodiesel production, is associated with some problems such as immiscibility with oils and lipase deactivation. To overcome these barriers, ethyl acetate was proposed as an alternative acyl acceptor for the production of biodiesel from soybean oil using an immobilized lipase, Novozym 435, Ethyl acetate mixed well with soybean oil, and only slightly inhibited the lipase activity by 5%. The effects of various environmental parameters, such as the composition of soybean oil and ethyl acetate, lipase content, and reaction temperature, were investigated to determine the optimal conditions for biodiesel production. As a result, the highest biodiesel production yield, 63.3 (±0.6)%, was obtained by using an ethyl acetate and soybean oil mixture with a 6∶1 molar ratio, with 8% of the immobilized lipase based on the weight of oil added at 70°C and 600 rpm.     

Mechanism of the polymerization reaction initiated and catalyzed by the polyhydroxybutyrate synthase of Ralstonia eutropha

Polyhydroxybutyrate (PHB) synthases (polymerases) catalyze the polymerization of the coenzyme A thioester of 3-hydroxybutyrate to PHB. The Ralstonia eutropha PHB synthase purified from recombinant E. coli cells exists in aqueous solution in both monomeric (single subunit) and homodimeric (two subunits) forms in equilibrium. Several lines of evidence suggest that the homodimer is the active form of the synthase. The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site. The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis, but a second thiol group on the same protein molecule has not yet been identified. It is suggested that cysteines at 319 from each of the two molecules of a homodimer synthase provide two identical thiol groups to jointly form a single catalytic site. To verify this model using the strategy of in vitro reconstitution, heterodimers composed of the wild-type subunit and of the C(319) mutated subunit were constructed and the activities at various ratios of the wild-type subunit to the mutated subunit were measured. The experimental results indicate that the homodimer is the active form of the enzyme, that the heterodimer containing the mutated subunit has no activity, and that a single cysteine is not sufficient for catalysis. Two identical thiol groups from C(319) residues on each subunit of the homodimer are required to form the catalytic site for the initiation and propagation reactions. We further demonstrate that a dimer synthase that has initiated the polymerization reaction (primed synthase) is significantly more stable against dissociation than the unprimed (unreacted) dimer synthase. These two properties explain the nature of lag phenomenon during the in vitro polymerization reaction catalyzed by this enzyme     

A novel transesterification catalyzed by phosphotriesterase in an organic solvent

Summary Phosphotriesterase (PTE) from Flavobacterium sp. was utilized for transesterification of the organophosphate insecticide, Paraoxon, using 2-phenylethyl alcohol as a nucleophile and dimethly sulfoxide as an organic solvent forming the converted phosphotriester compound, diethyl 2-phenylethyl phosphate.     

Mechanistic limitations in the synthesis of polyesters by lipase-catalyzed ring-opening polymerization

Lipase-catalyzed polymerization of caprolactone (CL) in toluene with methoxy-poly(ethylene glycol) (MPEG) and water as initiators was characterized in detail for mechanistic insight. (1)H NMR analysis of polycaprolactone chains (PCL), dicaprolactone, degree of esterification of MPEG, and fractions of PCL chains initiated by MPEG and water were used to follow the reactions. The data were analyzed with the kinetic scheme involving formation of the acylenzyme and its consequent reaction with MPEG, water, or PCL to yield the MPEG- or water-initiated PCL chains, or increase in PCL length. A limit for MPEG initiator esterification in lipase-catalyzed CL polymerization was observed and was explained by preferential reaction of PCL propagation over MPEG esterification at long reaction times and low MPEG concentrations. Slower monomer conversion in concentrated monomer solutions was explained by decreased partitioning of PCL between the solvent and the enzyme. This effect resulted in inhibition of the lipase by the reaction product, PCL chains, and/or insufficient diffusion of monomer to the enzyme active site. High monomer/initiators ratio in these solutions did not yield longer polymer chains due to decreased monomer conversion and the corresponding decrease in product yields; lower yields were also observed for chain initiation by MPEG and water. A shift in the reaction rate-limiting step from formation of acylenzyme in dilute CL solutions to its deacylation in concentrated CL solutions yielded higher PCL polydispersity due to increased initiation by water. Enhanced intramolecular cyclization was also observed. Endgroup composition of PCL chains was influenced by the concentration of monomer, ratio of initiators (MPEG and water), and reaction time, yielding PCL chains initiated exclusively by MPEG at "infinite reaction times."     

Enzymatic synthesis of polythioester by the ring-opening polymerization of cyclic thioester

A high molecular weight aliphatic polythioester was prepared by lipase-catalyzed polymerization of hexane-1,6-dithiol and dimethyl sebacate using the technique of ring-opening polymerization of a cyclic thioester. The cyclic thioester monomer was first prepared using lipase from Candida antarctica in dilute solution. The monomer was then polymerized by the same lipase in bulk to produce a polythioester with an M(w) of about 120 000 g/mol, which was significantly higher than that of a polythioester obtained by direct polycondensation of the dithiol and diacid. The polymerization rate and thermal properties of the product were measured and compared with those of the corresponding polyester prepared by ring-opening polymerization of a cyclic ester.     

Synthesis of poly(L-lactide) and polyglycolide by ring-opening polymerization

This protocol describes the synthesis of poly(L-lactide) by ring-opening polymerization of L-lactide using tin(II) 2-ethylhexanoate catalyst as well as the synthesis of polyglycolide by ring-opening polymerization of glycolide. Ring-opening polymerization of cyclic diesters synthesized from alpha-hydroxycarboxylic acids gives high-molecular-weight polyester in high yield. Tin(II) 2-ethylhexanoate catalyst is the most common catalyst for ring-opening polymerization of diesters owing to its high reactivity and low toxicity. Purity of monomers and the amount of water and alcohol in the reaction system are significant factors for increasing molecular weight and conversion of polyesters. The molecular weight of the polyesters is also dependent on reaction temperature and reaction time. This protocol can be completed in 3 d for the synthesis of poly(L-lactide) and 2 d for the synthesis of polyglycolide.     

Lipase catalyzed esterification of glycidol in organic solvents

We studied the resolution of racemic glycidol through esterification with butyric acid catalyzed by porcine pancreatic lipase in organic media. A screening of seven solvents (log P values between 0.49 and 3.0, P being the n-octanol-water partition coefficient of the solvent) showed that neither log P nor the logarithm of the molar solubility of water in the solvent provides good correlations between enantioselectivity and the properties of the organic media. Chloroform was one of the best solvents as regards the enantiomeric purity (e. p.) of the ester produced. In this solvent, the optimum temperature for the reaction was determined to be 35 degrees C. The enzyme exhibited maximum activity at a water content of 13 +/- 2% (w/w). The enantiomeric purity obtained was 83 +/- 2% of (S)-glycidyl butyrate and did not depend on the alcohol concentration or the enzyme water content for values of these parameters up to 200 mM and 25% (w/w), respectively. The reaction was found to follow a BiBi mechanism. (c) 1993 John Wiley & Sons, Inc.     

Lipase catalysis in organic solvents transesterification of O-formyl esters of secondary alcohols

Summary Behaviour of the O-formyl esters of sterically hindered racemic secondary alcohols towards lipase-catalysed transesterification in organic solvents was studied. They react about 40–60 times faster than their respective O-acetates.     

Biodiesel production by in situ transesterification of municipal primary and secondary sludges

The potential of using municipal wastewater sludges as a lipid feedstock for biodiesel production was investigated. Primary and secondary sludge samples obtained from a municipal wastewater treatment plant in Tuscaloosa, AL were freeze-dried and subjected to an acid-catalyzed insitu transesterification process. Experiments were conducted to determine the effects of temperature, sulfuric acid concentration, and mass ratio of methanol to sludge on the yield of fatty acid methyl esters (FAMEs). Results indicated a significant interactive effect between temperature, acid concentration, and methanol to sludge mass ratio on the FAME yield for the insitu transesterification of primary sludge, while the FAME yield for secondary sludge was significantly affected by the independent effects of the three factors investigated. The maximum FAME yields were obtained at 75 degrees C, 5% (v/v) H(2)SO(4), and 12:1 methanol to sludge mass ratio and were 14.5% and 2.5% for primary and secondary sludge, respectively. Gas chromatography (GC) analysis of the FAMEs revealed a similar fatty acid composition for both primary and secondary sludge. An economic analysis estimated the cost of $3.23/gallon for a neat biodiesel obtained from this process at an assumed yield of 10% FAMEs/dry weight of sludge.     

Synthesis of biodegradable poly-epsilon-caprolactone microspheres by dispersion ring-opening polymerization in supercritical carbon dioxide

A series of fluorinated diblock and triblock copolymers of poly(epsilon-caprolactone) and poly(heptadecafluorodecylacrylate) were prepared by combining ring-opening polymerization of epsilon-CL and atom transfer radical polymerization of the acrylate. These copolymers with well-controlled molecular weight and composition were characterized by (1)H NMR spectroscopy and used as stabilizers for the dispersion ring-opening polymerization of epsilon-CL in supercritical carbon dioxide. The effect of composition and architecture of the polymeric stabilizers on the stabilization of PCL microspheres was investigated. Finally, purification of PCL was successfully implemented by reactive supercritical fluid extraction of the tin catalyst.     

Regioselectivity of ferulic acid polymerization catalyzed by oxidases

Enzymatic oxidation of ferulic acid catalyzed by oxidases (laccase and peroxidase) was carried out. Ferulic acid was shown to be subjected to oxidative processes leading to the formation of oligomeric and polymeric structures. The polymer formation takes place due to the formation of C_Ar-C_Ar- and C_Ar-O-C_Ar-bonds, as well as due to reactions of opening of the propane chain double bond. Different dynamic conditions of the enzymatic reactions were used to study the effects of conditions on the biosynthesis in vitro of some dehydropolymers: the method of dropwise mixing (endwise polymers) and a single addition (bulk polymers). The chemical structures of the resulting compounds were examined by the methods of IR, ¹H NMR, and ¹³C NMR spectroscopy. Differences in the quantitative ratio of structural fragments in a polymer cause changes in its thermal characteristics.     

Asymmetric transesterification of secondary alcohols catalyzed by feruloyl esterase from Humicola insolens

A new asymmetric transesterification of secondary alcohols catalyzed by feruloyl esterase from Humicola insolens has been found. Although alcohols are not the natural substrates for this enzyme, a high R enantioselectivity was observed. Stereochemical studies showed that variations in substrate structure lead to strong variations in enantioselectivity. The highest enantioselectivities are obtained when the beta-carbon of the secondary alcohol is tertiary or quaternary.     

Regioselectivity of ferulic acid polymerization catalyzed by oxidases

Enzymatic oxidation of ferulic acid catalyzed by oxidases (laccase and peroxidase) was carried out. Ferulic acid was shown to be subjected to oxidative processes leading to the formation of oligomeric and polymeric structures. The polymer formation takes place due to the formation of CAr-CAr-, and CAr-O-CAr-bonds, as well as due to reactions of opening of the propane chain double bond. Different dynamic conditions of the enzymatic reactions were used to study the effects of conditions on the biosynthesis in vitro of some dehydropolymers: the method of dropwise mixing (endwise polymers) and a single addition (bulk polymers). The chemical structures of the resulting compounds were examined by the methods of IR, 1H NMR, and 13N NMR spectroscopy. Differences in the quantitative ratio of structural fragments in a polymer cause changes in its thermal characteristics.     

Enzymatic polymerization catalyzed by surfactant-coated lipases in organic media

Structural ring-opening of lactones driven by enzymatic polymerization has been performed using low concentration dosages of surfactant-coated lipases in organic media. By comparison, enzymatic polymerization rate with coated lipase proceeded at a rate 100-fold better than native powder. Similarly a higher polymeric molecular weight (21,300), narrow dispersity (Mw/Mn=1.9) and better conversion (100%) were obtained following polyesterification tests with surfactant-coated lipase.     

Potential biofuel additive from renewable sources--Kinetic study of formation of butyl acetate by heterogeneously catalyzed transesterification of ethyl acetate with butanol

Butyl acetate holds great potential as a sustainable biofuel additive. Heterogeneously catalyzed transesterification of biobutanol and bioethylacetate can produce butyl acetate. This route is eco-friendly and offers several advantages over the commonly used Fischer Esterification. The Amberlite IR 120- and Amberlyst 15-catalyzed transesterification is studied in a batch reactor over a range of catalyst loading (6–12 wt.%), alcohol to ester feed ratio (1:3 to 3:1), and temperature (303.15–333.15 K). A butanol mole fraction of 0.2 in the feed is found to be optimum. Amberlite IR 120 promotes faster kinetics under these conditions. The transesterifications studied are slightly exothermic. The moles of solvent sorbed per gram of catalyst decreases (ethanol > butanol > ethyl acetate > butyl acetate) with decrease in solubility parameter. The dual site models, the Langmuir Hinshelwood and Popken models, are the most successful in correlating the kinetics over Amberlite IR 120 and Amberlyst 15, respectively.