Lipase-catalyzed Reaction Research Articles

T enzymatic esterification of naringin and the properties of naringin esterified derivatization

As a kind of important natural compound with various physiological activities such as anti-oxidation, anti-inflammation, anti-aging, anti-cancer, anti-virus, etc, flavonoid had great application potential in food, cosmetics and pharmaceutical industries. However, the poor solubility limited their application. To solve this problem, one flavonoid glycoside, naringin, was esterified with lipase to improve its performance in the paper. Four fatty acids were selected as acyl donors to be esterified with naringin by single factor experiment, respectively. The results indicated that the optimal reaction conditions were the same: water activity was between 0.11 and 0.69, TLIM lipase was 50 mg/mL, acetone was used as the solvent, the molar ratio of substrate (naringin: fatty acid) was 1:10, and the substrates were first pretreated by ultrasound for 0.5 h, then reacted at 50 °C for 16 h. The conversion rates of naringin esterified with different fatty acids were all above 80.0%. Furtherly, the conversion rate of naringin was 93.9%, after the response surface experiment was carried out to optimize the lipase-catalyzed reaction conditions of naringin with palm oil. Lipophilicity and antioxidant ability of naringin esterified derivatives were measured. The results showed that the lipophilicity of naringin esterified derivatives was significantly higher than that of naringin. Although the free radical scavenging ability and reducing ability decreased by approximately 8% and 0.8%, respectively, the inhibiting oil peroxidation ability of naringin fatty acid esters were higher than that of naringin, which indicated that naringin esterified derivatives have the potential to become fat-soluble antioxidants.

Lipase-Catalyzed Kinetic Resolution of Dimethyl and Dibutyl 1-Butyryloxy-1-carboxymethylphosphonates

The main objective of this study is the enantioselective synthesis of carboxyhydroxyphosphonates by lipase-catalyzed reactions. For this purpose, racemic dimethyl and dibutyl 1-butyryloxy-1-carboxymethylphosphonates were synthesized and hydrolyzed, using a wide spectrum of commercially available lipases from different sources (e.g., fungi and bacteria). The best hydrolysis results of dimethyl 1-butyryloxy-1-carboxymethylphosphonate were obtained with the use of lipases from Candida rugosa, Candida antarctica, and Aspergillus niger, leading to optically active dimethyl 1-carboxy-1-hydroxymethylphosphonate (58%–98% enantiomeric excess) with high enantiomeric ratio (reaching up to 126). However, in the case of hydrolysis of dibutyl 1-butyryloxy-1-carboxymethylphosphonate, the best results were obtained by lipases from Burkholderia cepacia and Termomyces lanuginosus, leading to optically active dibutyl 1-carboxy-1-hydroxymethylphosphonate (66%–68% enantiomeric excess) with moderate enantiomeric ratio (reaching up to 8.6). The absolute configuration of the products after biotransformation was also determined. In most cases, lipases hydrolyzed (R) enantiomers of both compounds.

Micro-Aqueous Organic System: A Neglected Model in Computational Lipase Design?

Water content is an important factor in lipase-catalyzed reactions in organic media but is frequently ignored in the study of lipases by molecular dynamics (MD) simulation. In this study, Candida antarctica lipase B, Candida rugosa lipase and Rhizopus chinensis lipase were used as research models to explore the mechanisms of lipase in micro-aqueous organic solvent (MAOS) media. MD simulations indicated that lipases in MAOS systems showed unique conformations distinguished from those seen in non-aqueous organic solvent systems. The position of water molecules aggregated on the protein surface in MAOS media is the major determinant of the unique conformations of lipases and particularly impacts the distribution of hydrophilic and hydrophobic amino acids on the lipase surface. Additionally, two maxima were observed in the water-lipase radial distribution function in MAOS systems, implying the formation of two water shells around lipase in these systems. The energy landscapes of lipases along solvent accessible areas of catalytic residues and the minimum energy path indicated the dynamic open states of lipases in MAOS systems differ from those in other solvent environments. This study confirmed the necessity of considering the influence of the microenvironment on MD simulations of lipase-catalyzed reactions in organic media.

Preparation of acylated pectins with phenolic acids through lipase-catalyzed reaction and evaluation of their preservation performance

This study investigated the acylation of pectin with p-coumaric and caffeic acids through an enzymatic method to enhance its physical and chemical properties. Ultraviolet–visible, Fourier transform infrared and 1H nuclear magnetic resonance spectral analyses indicated the successful grafting of p-coumaric and caffeic acids onto the carboxyl group of pectin molecule. The 1,1-diphenyl-2-picryl hydrazine (DPPH) clearance and inhibition ratio in the β-carotene bleaching assay of native pectin (Na–Pe) were 0.13% and 42.60%, respectively, while their corresponding values increased to 1.04% and 55.59 for the pectin acylated with p-coumaric acid (Co–Pe) and 87.52% and 94.05% for the pectin acylated with caffeic acid (Ca–Pe). Meanwhile, the inhibition of Escherichia coli and Staphylococcus aureus was increased from 2.56% to 6.59% (Na–Pe) to 17.28% and 25.79% (Co–Pe) and 23.59% and 36.94% (Ca–Pe), respectively. Furthermore, Co-Pe or Ca-Pe coating was shown to significantly enhance the shelf life of small yellow croaker (Larimichthys polyactis) fillets.

Stability comparison of four lipases and catalytic mechanism during the synthesis of 1,3-di-oleic-2-medium chain triacylglycerols in a trace water-in-oil system: Experimental analyses and computational simulations.

In the present study, a kind of structured lipids, namely 1,3-di-oleic-2-medium chain (OMO) triacylglycerols, were synthesized through lipase-catalyzed reactions using coconut oil and rapeseed acid as materials in a trace water-in-oil system. Experimental analysis and computational simulations were undertaken to compare the stability of four lipases including Lipozyme RMIM, Lipozyme TLIM, Novozym 435, and Aspergillus oryzae immobilized lipase (AOIM), and illustrate catalytic mechanism of Novozym 435 during the synthesis of OMO. Fourier transform infrared and molecular dynamics simulation results demonstrated that a decrease in ordered structure (α-helix and β-sheet) led to a reduction in enzyme activity. Compared with Lipozyme RMIM and Novozym 435, Lipozyme TLIM and AOIM exhibited better stability due to a short-chain lid in TLIM, which covers activity sites, and hydrogen bonds formed between activity center of AOIM and water. Among four lipases, AOIM exhibited best catalytic performance: a OMO yield of 30.7% at 3hr and a good stability of long term (48hr). Density functional theory results demonstrated that specifically, during the synthesis of OMO triacylglycerol, the addition of Novozym 435 (derived from Candida antarctica lipase B, CALB) substantially lowered reaction barriers (64.4KJ/mol with CALB vs. 332.7KJ/mol with no lipase), aiding in the generation of OMO because of the formations of transitional tetrahedral intermediates. A trace water-in-oil system was a green and efficient alternative for lipase-catalyzed production of OMO, and this study provided crucial insights into the stability/instability and catalytic mechanisms of lipase in the synthesis of structured lipids. PRACTICAL APPLICATIONS: We compared the stability of Lipozyme RMIM, Lipozyme 435, Lipozyme TLIM, and AOIM during the synthesis of the OMO triacylglycerols in a trace water-in-oil system, where exhibited a high catalytic activity of lipase in water-oil interface. AOIM had the highest stability and showed the best catalytic performance due to the formation of hydrogen bonds. Besides, for the first time, the transition tetrahedral structure was revealed in the enzymatic synthesis of medium- and long-chain triacylglycerols. This study provides a rational approach to compare lipase stability and a possible hint to choose appropriate enzyme in a specific catalytic condition.

One-pot production of butyl butyrate from glucose using a cognate \u201cdiamond-shaped\u201d E. coli consortium

Esters are widely used in plastics, textile fibers, and general petrochemicals. Usually, esters are produced via chemical synthesis or enzymatic processes from the corresponding alcohols and acids. However, the fermentative production of esters from alcohols and/or acids has recently also become feasible. Here we report a cognate microbial consortium capable of producing butyl butyrate. This microbial consortium consists of two engineered butyrate- and butanol-producing E. coli strains with nearly identical genetic background. The pathways for the synthesis of butyrate and butanol from butyryl-CoA in the respective E. coli strains, together with a lipase-catalyzed esterification reaction, created a “diamond-shaped” consortium. The concentration of butyrate and butanol in the fermentation vessel could be altered by adjusting the inoculation ratios of each E. coli strain in the consortium. After optimization, the consortium produced 7.2 g/L butyl butyrate with a yield of 0.12 g/g glucose without the exogenous addition of butanol or butyrate. To our best knowledge, this is the highest titer and yield of butyl butyrate produced by E. coli reported to date. This study thus provides a new way for the biotechnological production of esters.

Surfactant Monolayer Bending Elasticity in Lipase Containing Bicontinuous Microemulsions.

Lipase-catalyzed reactions offer many advantages among which a high degree of selectivity combined with the possibility to convert even non-natural substrates are of particular interest. A major drawback in the applicability of lipases in the conversion of synthetically interesting, non-natural substrates is the substantial insolubility of such substrates in water. The conversion of substrates, natural or non-natural, by lipases generally involves the presence of a water–oil interface. In the present paper, we exploit the fact that the presence of lipases, in particular the lipase from Candida antarctica B (CalB), changes the bending elastic properties of a surfactant monolayer in a bicontinuous microemulsion consisting of D2O/NaCl -n-(d)-octane-pentaethylene glycol monodecyl ether (C10E5) in a similar manner as previously observed for amphiphilic block-copolymers. To determine the bending elastic constant, we have used two approaches, small angle neutron scattering (SANS) and neutron spin echo (NSE) spectroscopy. The time-averaged structure from SANS showed a slight decrease in bending elasticity, while on nanosecond time scales as probed with NSE, a stiffening has been observed, which was attributed to adsorption/desorption mechanisms of CalB at the surfactant monolayer. The results allow to derive further information on the influence of CalB on the composition and bending elasticity of the surfactant monolayer itself as well as the underlying adsorption/desorption mechanism.

Investigation on enzymatic activity of rubber seed as source of plant lipase

Rubber seed is a non-edible seed that is abundantly available and considers agricultural wastes. A potential lipase from rubber seed was examined based on the enzymatic activity and its application in the hydrolysis reaction. The enzymatic activity characterization study was determined based on p-nitrophenol release in the hydrolysis reaction. The initial evaluation showed that temperature and pH significantly influence the reaction. The optimum condition based on enzymatic activity for rubber seed was found at 30 ℃ and pH 8. The rubber seed lipase extract was then used in enzymatic hydrolysis reactions of rubber seed oil, palm oil, and canola oil. The highest FFA percentage of 63% was found from the rubber seed oil. The results indicate that rubber seed extract has shown its potential enzymatic activity. However, further studies need to be done to apply this rubber seed in various lipase catalysed reactions.

Investigation of a Lipase-Catalyzed Reaction between Pectin and Salicylic Acid and Its Isomers and Evaluation of the Emulsifying Properties, Antioxidant Activities, and Antibacterial Activities of the Corresponding Products.

This study presents a method for modifying pectin with phenolic acids catalyzed by lipase in a two-phase system of water/tetrahydrofuran. Salicylic acid (SA) and its isomers, including m-hydroxybenzoic acid (MHBA) and p-hydroxybenzoic acid (PHBA), were grafted onto pectin, and the products were characterized via UV-vis, Fourier transform infrared spectroscopy (FTIR), and 1H NMR analyses to explore the reaction process and mechanism between pectin and the three phenolic acids. Results indicated that lipase played a dual role in the reaction, namely, catalyzing the hydrolysis of the methyl group in the aqueous phase and esterifying the carboxyl group of pectin with the phenolic hydroxyl group of the phenolic acids in tetrahydrofuran. The grafting ratio of SA-modified pectin, MHBA-modified pectin, and PHBA-modified pectin was 1.89, 10.58, and 20.32%, respectively, and it was affected by the position of phenolic hydroxyl. Moreover, the effects of phenolic acids on the emulsifying properties, antioxidant activities, and antibacterial activities of the native and modified pectins were evaluated. In several aspects, the emulsifying properties of the modified pectins were better than those of native pectin. Moreover, the grafting of phenolic acids only slightly affected the 1,1-diphenyl-2-picryl hydrazine (DPPH) clearance of the modified pectins but substantially improved their inhibition ratio in a β-carotene bleaching assay. Furthermore, the modified pectins exhibited better bacteriostatic activity against both Escherichia coli and Staphylococcus aureus than native pectin.

Reaction kinetic studies on the immobilized-lipase catalyzed enzymatic resolution of 1-phenylethanol transesterification with ethyl butyrate

The enzyme-catalyzed transesterification reaction is an important route to realize the resolution of racemic optically active secondary alcohols. However, this route suffers from the high environment and engineering risks due to the usage of vinyl ester as acyl donor. In this work, an alternative acyl donor, ethyl butyrate, was used because it is more environment-friendly and catalyst-friendly. The kinetic studies on transesterification of 1-phenylethanol with ethyl butyrate catalyzed by immobilized-lipase Novozym™ 435 were carried out. The effects of agitation speed, reaction temperature, catalyst loading and initial molar ratio of reactants were investigated. The Ping-Pong-Bi-Bi and pseudo-homogeneous models were used to correlate the experimental data to estimate the kinetic parameters. The concentration-based Ping-Pong-Bi-Bi model was translated into its activity-based form to describe the non-ideality of the lipase-catalyzed reaction system. The activity-based Ping-Pong-Bi-Bi kinetic model performed best among all the models used.

Polyamidoamine dendrimers: Favorable polymeric nanomaterials for lipase activation

Dendrimers are highly branched synthetic macromolecules that offer promising prospects for utilizing them in broad applications. This study investigates the role of polyamidoamine (PAMAM) dendrimer for lipase-catalyzed reaction in aqueous solution. The hydrolysis of para-nitrophenyl palmitate (pNPP) by four different lipases was tested with PAMAM dendrimer with different generations (i.e., D0.0, D2.5, D3.0, and D4.0). The results showed that the activity of Rhizopus niveus lipase (RNL) was enhanced up to 375 % with PAMAM dendrimer (D0.0). The optimum incubation conditions such as the temperature and coupling time of the lipase with D0.0 were 40 °C and 2 h, respectively. The behavior of lipases at different pH showed that RNL was mostly active at pH 7.0. Most lipases with D0.0 were enhanced at least two-fold compared to the control samples. Kinetic study showed that the RNL affinity was enhanced in the presence of PAMAM. FTIR spectra strongly suggest that hydrogen bonding and electrostatic interactions were the main types of bonding in the RNL and PAMAM system. Additionally, the molecular docking simulation of the system was exerted to evaluate and support the related compound, (i.e., D0.0) as a ligand interacted effectively with RNL based on the experimental activity. The cytotoxicity study revealed mild toxicity of the system which could serve as a potential target in nanomedicine for many applications such as cancer therapeutics.

Greener approach for synthesis of N,N,N-trimethyl chitosan (TMC) using ternary deep eutectic solvents (TDESs)

N,N,N-trimethyl chitosan (TMC), quaternized hydrophilic derivative of chitosan, has been projected to have wide applications in the pharmaceutical industry owing to its improved solubility at physiological conditions. However, the conventional synthesis of TMC involves toxic organic agents, which complicates its use for biological applications. Moreover, these reactions result into unwanted O-methylation and scission of the parent polymer. In the present study we have addressed these limitations by employing a green approach to synthesize TMC, by using lipase as the biocatalyst and dimethyl carbonate (DMC) as the green methylating agent, in a reaction medium comprising of ternary deep eutectic solvents (TDESs). Synthesis of TMC was carried out by using two different lipases from Burkholderia cepacia and Candida rugosa. The resulting TMC was characterized by using FTIR, 1H NMR, DSC, XRD. Methylation was confirmed by FTIR analysis (-CH at 1666 cm−1) and 1H NMR (?? = 3.3 ppm). DSC study revealed a lower thermal stability of TMC as compared to chitosan. These results indicated the possibility of using DMC as a green methylating agent, along with TDESs as green and sustainable solvents, for lipase catalyzed reactions. TMC was successfully synthesized and exhibited a degree of quaternization of about 12.5%, 15.69%, when synthesized used lipases from Burkholderia cepacia and Candida rugosa, respectively.

Deep eutectic solvents for biocatalytic transformations: focused lipase-catalyzed organic reactions

Biocatalysis is a green and sustainable technology for which the ideal solvent should be nontoxic, biocompatible, biodegradable, and sustainable, in addition to supporting high enzyme activity and stability. Deep eutectic solvents (DESs), a novel class of green solvents, have recently emerged as excellent alternatives for use in various biocatalytic reactions and, in particular, in lipase-catalyzed reactions with enzymes. This review discusses the achievements that have been made so far in the use of DESs as reaction media for lipase-catalyzed reactions. In addition, the application of DESs in esterification, transesterification, and amidation reactions with isolated or immobilized biocatalysts, toward enabling the synthesis of biodiesels, sugar esters, phenolipids, and fatty acyl ethanolamides, is summarized, while advances in lipase-catalyzed chemoenzymatic epoxidation reactions, C-C bond-forming Aldol reactions, and hydrolysis reactions in DESs are also discussed. This review also summarize some remaining questions concerning the use of DESs, including the intriguing role of water as a cosolvent in biocatalytic reactions carried out in DESs, and the relationship between the nature of the DESs and their influence on the enzyme stability and activity at the molecular level.

Efficient Synthesis of Bio-Surfactant from D-Glucose by Immobilized Candida cylindracea Lipase-Catalyzed Reaction in Ionic Liquids

Efficient Synthesis of Bio-Surfactant from D-Glucose by Immobilized Candida cylindracea Lipase-Catalyzed Reaction in Ionic Liquids

Biodiesel-mediated biodiesel production: A recombinant Fusarium heterosporum lipase-catalyzed transesterification of crude plant oils

Production of biodiesel from unrefined plant-derived oils has been considerably investigated but, to date, there appears to be a limit by phospholine gums and other contaminants in its conversion to biodiesel. The presence of such contaminants increases the melting point of the oils, thereby, making it unfavorable for lipase-catalyzed reactions at mild temperatures. Here, to circumvent this limiting effect, we explore the potential of biodiesel as a replacement for conventional organic solvents in solubilizing gum-formation contaminants in crude palm oil (CPO). A strategy of 1:1 CPO/biodiesel molar amount consolidates degumming and trans/esterification into one-step using immobilized Aspergillus oryzae expressing Fusarium heterosporum lipase. The innocuous biodiesel solvent not only ensured a 98.8 wt% biodiesel yield but also improved the catalytic activity of the whole-cell lipase. This allowed repeated use of the recombinant lipase in nine consecutive batches. At room temperature, biodiesel as a solvent saves on post-separation and reduces environmental footprints of the biodiesel production process.

Physical and emulsion stabilizing properties of maltodextrin fatty acid polymers produced by lipase-catalyzed reactions in ethanol

Maltodextrin (MD) fatty acid esters (MFAs) have amphiphilic properties and the enzymatic synthesis of these molecules has gained growing interest. Here, MFAs were synthesized in a food-grade ethanol system and the properties of the products were analyzed. A total of 6 different MFAs were produced with 2 different MD sources and 3 combinations of fatty acids (lauric, palmitic, and both) with yields ranging from 72.7 to 83.4%. With an increase in fatty acid carbon length, degree of substitution (0.026 to 0.016) and solubility (100.9% to 93.1%) were significantly decreased. The stability of emulsions formulated with MFAs was investigated and all emulsions formulated were stable except those containing the lowest concentration of MFAs esterified with palmitate. Notably, MD esterified with laurate showed an enhanced emulsion stabilizing ability as compared to commercial emulsifiers. In conclusion, the emulsion stabilizing ability of MFAs may have applications in the food industry.

LIPASE - CATALYZED FORMATION OF PENTYL NONANOATE USING SCREENED IMMOBILIZED LIPASE FROM Rhizomucor meihei

Abstract Bio-catalysis has attracted the special attention of industrial flavour producers in the production of valuable ester compounds. In this study, the synthesis of pentyl nonanoate ester (a short chain ester with fruity aroma) was carried out with a commercial immobilized lipase from Rhizomucor meihei Lipozyme (RMIM) as biocatalyst in the esterification reaction between nonanoic acid and pentanol. Various reaction parameters such as enzyme concentration, substrate concentration, reaction temperature and reaction time in solvent-free system were screened to enhance the ester formation with the best yield. A maximum yield for pentyl nonanoate (86.08 %) in a solvent-free system was obtained within 150 min, at a reaction temperature of 45 OC, molar ratio of 1:9 M, amount of enzyme of 0.2 g, water addition of 0.2 % v/v and shaking speed of 150 rpm. This work suggests that pentyl nonanoate ester can be produced in a very high yield and in a short period by lipase-catalysed reactions of nonanoic acid and pentanol, using immobilized lipase from RMIM (lipase from Rhizomucor miehei immobilized on anionic exchange support).

Biocatalytic synthesis and characterization of sn-1/3 and sn-2 monoacylglycerols.

To investigate the lipase-catalyzed synthesis of high purity sn-1/3 and sn-2 monoacylglycerols (1/3-MAG and 2-MAG) of different fatty acids (FAs). The 1/3-MAGs of three FAs (16:0, 17:0, 16:1) were synthesized using lipase-catalyzed esterification of glycerol with FAs. The 2-MAGs were obtained from the ethanolysis of synthetic triacylglycerols using sn-1,3 regiospecific lipase. The effects of lipase types, substrate ratio, temperature, reaction time and lipase load on the MAG conversion were studied. Under the optimal conditions, high purities (96.74%, 95.44%, 92.96%) with acceptable isolated yields (51.00%, 54.28%, 46.00%) were obtained for 1/3-16:0-MAG, 1/3-17:0-MAG, and 1/3-16:1-MAG, respectively. For 2-16:0-MAG, 2-17:0-MAG, and 2-16:1-MAG, the purities were 92.64, 95.04, and 96.48%, with isolated yields of 50.64, 52.16, and 26.12%, respectively. The molecular structures of the synthetic compounds were confirmed by 1H NMR, and MS and the melting points were characterized by DSC. High purity MAG isomers can be synthesized via lipase-catalyzed reactions to be building blocks for production of functional lipids, the melting points of which are largely governed by the hydrophobic interactions among the alkanyl chains.

'Clean' hydrolase reactions using commercial washing powder.

We report the use of commercial laundry powder as a biocatalyst for a range of lipase-catalysed reactions including (trans)esterification, ester hydrolysis and chemoenzymatic epoxidation reactions. The enzymatic laundry powder exhibited excellent stability and recyclability, making it a readily available and cheap biocatalyst for chemical transformations.

Microbial biodiesel production: From sucrose-based carbon sources to alkyl esters via enzymatic transesterification

Abstract Sucrose, in contrast to glucose, is a lower-cost carbon source. This manuscript discloses that its use in submerged fungal growth promotes lipids more suitable to biodiesel production due to lower unsaturation indexes. The wild strain of Mucor circinelloides URM 4182, a potential source of microbial lipid, was grown on three sucrose-based carbon sources: commercial brown sugar, sugarcane juice, and sugarcane syrup, achieving biomass accumulation productivities up to 3.62 ± 0.04 g L−1 day−1, and lipid productivities as high as 0.93 ± 0.01 g L−1 day−1. Lipid extraction was performed using ethanol, a green alternative to petroleum-derived solvent, and characterized according to their fatty acid distribution, from which C16:0 and C18:1 stand as the most abundant fatty acids. Extracted lipids were converted to ethyl esters via lipase-catalyzed reactions achieving ester contents up to 97.4% and low contents of mono- and diacylglycerols (1.9% and 1.0%, respectively). The interest in utilizing sucrose-based sources is due to the wide abundance of such feedstock in sugarcane-producing regions, and due to the potential of increasing value to these low-cost carbon sources. The results presented herein demonstrate a simple, yet efficient, process of converting sucrose and its derivatives to microbial biodiesel using green-chemistry processes, such as lipid extraction using ethanol, avoiding the use of harsh and toxic chemicals commonly utilized, and enzyme-catalyzed reactions.