Lipase-catalyzed polymerization of lactones and linear hydroxyesters

Ring-opening polymerization of six lactones and condensation polymerization of six linear hydroxyesters were carried out at 45°C using a lipase from Pseudomonas sp. Ring-opening polymerization of the lactones gave both higher molecular weight and higher monomer conversion than condensation of corresponding linear hydroxyesters. Lipase-catalyzed copolymerization of -caprolactone with lactide and cyclopentadecanolide gave higher molecular weight compared with copolymerization of -caprolactone with corresponding linear hydroxyesters. © Rapid Science Ltd. 1998     

Lipase/esterase-catalyzed synthesis of aliphatic polyesters via polycondensation: A review

Over the last decade, there has been an increasing interest in lipase/esterase-catalyzed polycondensation as an alternative to metal-based catalytic process, because the former can proceed under mild reaction conditions and does not cause undesirable side reactions or produce trace metallic residues. In this review, the in vitro synthesis of aliphatic polyesters by polycondensation using lipases or esterases is systematically summarized, especially for the synthesis of complex and well-defined polyesters. The polycondensation of diols with diacids or their activated esters, including alkyl, haloalkyl and vinyl esters, through esterification and transesterification polycondensation reactions is discussed. In addition, three or more monomers can also be polymerized simultaneously, which provides a new route for preparing functional polymers. Self-polycondensation with respect to hydroxyl and mercapto acids or their esters is another reaction mode discussed in the review. Finally, concurrent enzymatic ring-opening polymerization and polycondensation has been developed to construct novel polyesters with tailor-made structures and properties. Overall, the review demonstrates that lipase/esterase-catalyzed synthesis of polyesters via polycondensation provides an effective platform for conducting “eco-friendly polymer chemistry”.     

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.     

Lipase catalyzed HEMA initiated ring-opening polymerization: in situ formation of mixed polyester methacrylates by transesterification

2-Hydroxyethyl methacrylate (HEMA) was used as initiator for the enzymatic ring-opening polymerization (ROP) of omega-pentadecalactone (PDL) and epsilon-caprolactone (CL). The lipase B from Candida antarctica was found to catalyze the cleavage of the ester bond in the HEMA end group of the formed polyesters, resulting in two major transesterification processes, methacrylate transfer and polyester transfer. This resulted in a number of different polyester methacrylate structures, such as polymers without, with one, and with two methacrylate end groups. Furthermore, the 1,2-ethanediol moiety (from HEMA) was found in the polyester products as an integral part of HEMA, as an end group (with one hydroxyl group) and incorporated within the polyester (polyester chains acylated on both hydroxyl groups). After 72 h, as a result of the methacrylate transfer, 79% (48%) of the initial amount of the methacrylate moiety (from HEMA) was situated (acylated) on the end hydroxyl group of the PPDL (PCL) polyester. In order to prepare materials for polymer networks, fully dimethacrylated polymers were synthesized in a one-pot procedure by combining HEMA-initiated ROP with end-capping using vinyl methacrylate. The novel PPDL dimethacrylate (>95% incorporated methacrylate end groups) is currently in use for polymer network formation. Our results show that initiators with cleavable ester groups are of limited use to obtain well-defined monomethacrylated macromonomers due to the enzyme-based transesterification processes. On the other hand, when combined with end-capping, well-defined dimethacrylated polymers (PPDL, PCL) were prepared.     

Lipase stereoselectivity and regioselectivity toward three isomers of dicaprin: A kinetic study by the monomolecular film technique

Here we present a kinetic study on the steroselectivity and regioselectivity of 23 purified lipases of animal and microbial origin. This work, concerning a general problem of the mechanism of lipase–substrate molecular recognition, was performed using pure dicaprin isomers: 1,2-sn-dicaprin, 2,3-sn-dicaprin, and 1,3-sn-dicaprin spread as monomolecular films at the air–water interface. The first two isomers are optically active antipodes (enantiomers), forming stable films up to 40 mN m^?1, while the last is a prochiral compound, with a surface pressure of collapse of 32 mN m^?1. To our knowledge, this is the first report on the use of three diglyceride isomers as lipase substrates under identical and controlled physicochemical conditions. The lipases tested display a typical behaviour, characteristic of each enzyme, which allowed us to classify the lipases in groups according to (1) the profiles of enzyme velocity as a function of surface pressure, (2) their preferences for a given diglyceride isomer, quantified using new parameters termed steroselectivity index (S.I.), vicinity index (V.I.), and surface pressure threshold (S.P.T.). The general observation, true for all the enzymes tested, is that the three substrates are well differentiated, and the differentiation is more pronounced at high interfacial energy (low surface pressure). This observation supports our hypothesis that lipase conformational changes, resulting from the enzymesurface interaction, affect the enzymes' specificities. Generally speaking, the stereopreference for either sn-1 or sn-3 position on glycerides is maintained both in the case of di- and tri-glycerides. © 1995 Wiley-Liss, Inc.     

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."     

From infinite tubular arrays to discrete molecules and back: An example of reversible ring-opening polymerization in silver(I)-diphosphazane chemistry

The reaction of AgX with the diphosphazane ligand, PrⁱN(PPh₂)₂ (L) gives the polymeric complexes, [Ag₂(μ-X)₂(μ-L)]_n (X = NO₃1a or OSO₂CF₃1b). Single crystal X-ray analysis of 1a reveals a novel structural motif formed by interlinking of giant 40-membered rings; the diphosphazane ligand L adopts a unique ‘C_s’ geometry. These polymeric complexes exhibit a completely reversible ring-opening polymerization-depolymerization relationship with the dinuclear and mononuclear complexes, [{Ag(μ-L)(X)}₂] (X = NO₃2a, X = OSO₂CF₃2b) and [Ag(κ²-L)₂]X (X = NO₃3a, X = OSO₂CF₃3b).     

Spectrophotometric Assay of Lipase Activity: A New 4-nitrophenyl Ester of a Dialkylglycerol Suitable as a Chromogenic Substrate of Pseudomonas cepacia Lipase

Evaluation of Pseudomonas cepacia lipase (PCL) activity by a titrimetric method with triacylglycerols (TAG) and synthetic dialkylglycerol esters (DAGE) established the chain length selectivity of the enzyme and this information has been used to design a new chromogenic substrate [1,2-di-O-octyl-sn-glycerol-3-O-(4-nitrophenyl) glutarate] for the determination of the lipolytic activity of PCL.     

Spectrophotometric Assay of Lipase Activity: A New 4-nitrophenyl Ester of a Dialkylglycerol Suitable as a Chromogenic Substrate of Pseudomonas cepacia Lipase

Evaluation of Pseudomonas cepacia lipase (PCL) activity by a titrimetric method with triacylglycerols (TAG) and synthetic dialkylglycerol esters (DAGE) established the chain length selectivity of the enzyme and this information has been used to design a new chromogenic substrate [1,2-di-O-octyl-sn-glycerol-3-O-(4-nitrophenyl) glutarate] for the determination of the lipolytic activity of PCL.     

Rapid functional identification of putative genes based on the combined in vitro protein synthesis with mass spectrometry: a tool for functional genomics

For the rapid identification of functional activity of unknown genes from a sequence database, a new method based on in vitro protein synthesis combined with mass spectrometry was developed. To discriminate their subtle enzymatic activity, in vitro synthesized and one-step purified lipolytic enzymes, such as lip A and lip B from Bacillus subtilis and an unknown protein ybfF from Escherichia coli, were reacted with a mixture of triglycerides with different carbon chain lengths. Using direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of reaction product, all three enzymes were revealed to have strong esterase activity rather than true lipase activity, which has no reactivity on long-chain fatty acids such as triolein. These results were also confirmed by classical color assay using p-nitrophenyl butyrate (pNPB) and p-nitrophenyl palmitate (pNPP) as representative lipolytic substrates.     

Lipase catalysed synthesis of diacyl hydrazines: an indirect method for kinetic resolution of chiral acids

The acylation of hydrazine, to afford the N,N′-diacyl derivatives, was catalysed by a number of lipases. The rates of the first and second steps depended on the lipase and the type of solvent used. Water, up to 0.4 M, had no detrimental effect on the yield and complete conversion to the N,N′-diacyl derivative was accomplished with some lipases. The hydrazide of 2-(4-isobutylphenyl)propanoic acid (ibuprofen), prepared by non-enzymatic reaction of ibuprofen methyl ester with hydrazine, acted as a nucleophile towards several lipases that do not accept ibuprofen derivatives as the acyl donor, but the enantiomer differentiation was inefficient in most cases. The best result was obtained with Pseudomonas lipoprotein lipase on EP 100 which formed the (R) enantiomer of the product (N-octanoyl-N′-2-(4-isobutylphenyl)propanoylhydrazine) with an enantiomeric ratio E of 26.     

The Mechanisms of Action and Inhibition of Pancreatic Lipase and Acetylcholinesterase: A Comparative Modeling Study

Abstract

Pancreatic lipase and acetylcholinesterase are both serine esterases. Their X-ray structures reveal a similar overall fold, but no sequence homology can be detected. A catalytic triad like in the trypsin family of serine proteases consisting of serine, histidine and aspartate (glutamate in acetylcholinesterase) suggests mechanistic similarities. Models of the transition states of the substrate cleavage have been built and possible catalytic pathways were examined. The model that could produce a consistent pathway throughout the reactions had a transition state of the opposite handedness compared to trypsin. These models could be used to rationalise binding modes of inhibitors of both enzymes. The lipase inhibitor tetrahydrolipstatin (THL) contains a gamma-lactone which is opened by the catalytic serine; the alcohol leaving group prohibits deacylation by locking the pathway for incoming water and thus inactivates the enzyme. Carbamate inhibitors of acetylcholinesterase transfer a carbamoyl group to the serine-OH which deacylates slowly. These observations can be used as a starting point for the discovery of new classes of inhibitors.     

黑曲霉脂肪酶：基因克隆、表达及体外复性

根据黑曲霉F044脂肪酶N-端氨基酸序列,运用生物信息学方法,找到与黑曲霉脂肪酶基因同源的候选基因A84689。根据该基因序列,设计引物直接PCR扩增得到黑曲霉脂肪酶全长基因anl。anl全长1044bp,含3个内含子,编码297个氨基酸(含信号肽27个氨基酸),与其它脂肪酶基因没有明显同源性。将编码成熟脂肪酶的anl连接到pET28a载体上得到重组表达质粒,转化大肠杆菌BL21(De3),诱导表达并纯化出目的蛋白。通过大量稀释和DEAESepharoseFastFlow层析相结合的方法,变性后的纯化蛋白在体外实现再折叠复性。     

A simple enzymatic procedure for the synthesis of a hydroxylated polyester from glycerol and adipic acid

Lipase B from Candida antarctica catalyzed regioselectively the polyesterification of glycerol and adipic acid. UV-MALDI-TOF-MS analysis of the polymers shows low molecular weight polyesters (1314-1716) with very narrow polydispersities (1.0–1.2).     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.     

Lipase immobilized in ordered mesoporous silica: A powerful biocatalyst for ultrafast kinetic resolution of racemic secondary alcohols

Although Burkholderia cepacia lipase (BCL) has been proved to be a potential catalyst for chiral resolution, it is rarely applied in industry because of the low catalysis activity and poor stability of the free form. In this article, BCL was immobilized on the phenyl-modified ordered mesoporous silica (Ph-OMMs) to obtain a novel immobilized lipase. Benefits from the bottle-neck mesoporous structure, high loading of BCL could be completed within only 15 min. When BCL@Ph-OMMs was used as a catalyst for the resolution of 1-phenylethanol, up to 50% conversion with more than 99% ee_s was obtained within only 25 min, which is about 65-folds faster than that of the free lipase. Stabilized BCL@Ph-OMMs was successfully used for the ultrafast resolution of six secondary alcohols by selectivity transesterification, which reached high conversion (50%) and high enantioselectivity (≥99%) within 20–180 min. The activity of BCL@Ph-OMMs was kept relatively constant in 50 consecutive cycles, which is the best result among the reported immobilized lipases. The study suggests that BCL@Ph-OMMs is an attractive catalyst in industrial applications.     

Immobilized Candida antarctica lipase B: Hydration, stripping off and application in ring opening polyester synthesis

This work reviews the stripping off, role of water molecules in activity, and flexibility of immobilized Candida antarctica lipase B (CALB). Employment of CALB in ring opening polyester synthesis emphasizing on a polylactide is discussed in detail. Execution of enzymes in place of inorganic catalysts is the most green alternative for sustainable and environment friendly synthesis of products on an industrial scale. Robust immobilization and consequently performance of enzyme is the essential objective of enzyme application in industry. Water bound to the surface of an enzyme (contact class of water molecules) is inevitable for enzyme performance; it controls enzyme dynamics via flexibility changes and has intensive influence on enzyme activity. The value of pH during immobilization of CALB plays a critical role in fixing the active conformation of an enzyme. Comprehensive selection of support and protocol can develop a robust immobilized enzyme thus enhancing its performance. Organic solvents with a log P value higher than four are more suitable for enzymatic catalysis as these solvents tend to strip away very little of the enzyme surface bound water molecules. Alternatively ionic liquid can work as a more promising reaction media. Covalent immobilization is an exclusively reliable technique to circumvent the leaching of enzymes and to enhance stability. Activated polystyrene nanoparticles can prove to be a practical and economical support for chemical immobilization of CALB. In order to reduce the E-factor for the synthesis of biodegradable polymers; enzymatic ring opening polyester synthesis (eROPS) of cyclic monomers is a more sensible route for polyester synthesis. Synergies obtained from ionic liquids and immobilized enzyme can be much effective eROPS.     

Structure, function and interfacial allosterism in phospholipase A2: insight from the anion-assisted dimer

Enzymes that function on membrane surfaces offer many challenges to understanding structural and functional details due to the difficulties of obtaining relevant information of the protein in a physiological environment. Focusing on this aspect of structural biology, it is important to develop conditions that mimic the interaction of membrane proteins with their binding surface and ultimately the mechanisms of action. This approach has been used to characterize the allosteric nature of secreted phospholipase A2 (PLA2) to its substrate interface. The breakthrough here was to crystallize the pancreatic group-IB PLA2 in an anion-assisted dimer with five coplanar phosphate anions bound. In the anion-assisted dimer structure one molecule of a tetrahedral mimic inhibitor and five anions are shared between the two subunits of the dimer. The sn-2-phosphate of the inhibitor, which mimics the tetrahedral intermediate of the esterolysis reaction, is bound in the active site of one subunit, and the alkyl chain extends into the active site slot of the second subunit across the subunit-subunit interface. This interface-bound structural mimic provided insight into the active site environment and specific anionic interactions to the i-face of the protein. The presence or absence of a single critical active site water, corresponds to the difference between the activated or inactivated form of the enzyme. The anion-assisted dimer structure supports a calcium coordinated nucleophilic water mechanism, with its pK(a) modulated by this assisting water. This working model has been further strengthened with an enzyme-product complex structure solved with the hydrolysis products of the substrate PAF also bound to the anion-assisted dimer form of PLA2. Additional confirmation of the assisting-water mechanism comes from a structure of the inactive zymogen proPLA2 also crystallized in an anion-assisted dimer. Remarkably, the assisting water present in the activated complex is absent in this proPLA2 structure.     

Enzymatic synthesis of short-chain diacylated alkylglycerols: A kinetic study

Lipase-catalyzed transesterification of 1-O-octadecyl glycerol (batyl alcohol) with ethyl butyrate has been studied. The effect of vacuum on the rate of both transesterification and distillation of ethyl butyrate has also been investigated and at different reaction conditions, more than 85% of 1-O-octadecyl glycerol was consumed in only 5 min giving rise to the monoester. Then the monoester is again acylated to produce the diesterified product. In addition, the transesterification reactions were effected in solvent free reaction medium and it has been scaled-up to produce up to ca. 500 g of 2,3-dibutyroil-1-O-alkylglycerols in three consecutive cycles reutilizing the same batch of lipase. An efficient evaporation of ethanol was necessary to significantly reduce the reaction times of the transesterification reaction. Finally, a kinetics model describing both the rate of transesterification and the rate of inactivation of the immobilized lipase has been developed. The results indicate that the operational stability of the immobilized lipase confined into the mesh baskets (according to the value of k_d attained), was very high and that provides a half-life of the lipase higher than 1500 h.The present procedure is intended to be used for the synthesis of homogeneous alkylglycerols with biological activities and/or precursors of structured alkylglycerols.