Immobilized MAS1 Lipase-catalyzed Synthesis of n-3 PUFA-rich Triacylglycerols in Deep Eutectic Solvents.

n-3 polyunsaturated fatty acids (PUFA)-rich triacylglycerols (TAG) with many beneficial effects are still difficult to be synthesized efficiently and rapidly by current synthetic techniques. This study reports the fatty acid specificity of immobilized MAS1 lipase and its efficient synthesis of n-3 PUFA-rich TAG by esterification of glycerol with n-3 PUFA in natural deep eutectic solvents (NADES) systems. Immobilized MAS1 lipase showed the highest preference for capric acid [C10:0, the highest specificity constant (1/α)=1] whereas it discriminated strongly against docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) due to their lowest specificity constants (1/α=0.19 and 0.2). Moreover, the highest n-3 PUFA-rich TAG content (55.8%) with similar n-3 PUFA composition to the substrate was obtained in choline chloride/glycerol (CG) system. There was a 1.38-fold increase of TAG content in CG system compared with that in the solvent-free system. Interestingly, immobilized MAS1 lipase exhibited no regiospecificity in the solvent-free and various NADES systems. Besides, the potential reaction mechanism of immobilized MAS1 lipase-catalyzed esterification of glycerol with n-3 PUFA in NADES systems was described. It was found that the use of NADES as solvents could greatly enhance TAG content, and make it easy to separate the product. These results indicated that immobilized MAS1 lipase is a promising biocatalyst for the efficient synthesis of n-3 PUFA-rich TAG by esterification of glycerol with n-3 PUFA in NADES systems.

Properties of immobilized MAS1-H108A lipase and its application in the efficient synthesis of n-3 PUFA-rich triacylglycerols.

This study reports the properties of immobilized MAS1-H108A lipase from marine Streptomyces sp. strain W007 on XAD1180 resin and its application in the synthesis of n-3 polyunsaturated fatty acids (PUFA)-rich triacylglycerols (TAG) for the first time. It was found that the optimal temperature and pH for both immobilized MAS1-H108A lipase and free lipase MAS1-H108A were 70 °C and 7.0, respectively. However, immobilized MAS1-H108A lipase exhibited higher thermostability when compared with free lipase MAS1-H108A. It was also interesting that both immobilized MAS1-H108A lipase and free lipase MAS1-H108A showed no regiospecificity in the hydrolysis of triolein. Subsequently, immobilized MAS1-H108A lipase and free lipase MAS1-H108A were employed to catalyze glycerolysis of n-3 PUFA-rich ethyl esters (EE) and esterification of n-3 PUFA with glycerol under vacuum in the solvent-free system. The results showed that n-3 PUFA-rich TAG were synthesized efficiently by non-regiospecific immobilized MAS1-H108A lipase and TAG contents separately reached 92.07% and 76.13% during the esterification and glycerolysis reactions, which were significantly higher than those (71.82% and 39.62%, respectively) obtained by free lipase MAS1-H108A. Besides, TAG exhibited similar n-3 PUFA composition to the substrate. These findings indicated that non-regiospecific immobilized MAS1-H108A lipase is a promising and efficient biocatalyst for the industrial synthesis of n-3 PUFA-rich TAG.

Synthesis and enantiospecific analysis of enantiostructured triacylglycerols containing n-3 polyunsaturated fatty acids.

The stereospecific structure of triacylglycerols (TAGs) affects the bioavailability of fatty acids. Lack of enantiopure reference compounds and effective enantiospecific methods have hindered the stereospecific analysis of individual TAGs. Twelve novel enantiostructured AAB-type TAGs were synthesized containing one of the three n-3 polyunsaturated fatty acid: α-linolenic acid (ALA), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) in sn-1 or sn-3 position. These compounds formed six enantiomer pairs, which were separated with recycling high-performance liquid chromatography using chiral columns and UV detection. The chromatographic retention behavior of the enantiomers and the stereospecific elution order were studied. The enantiomer with an n-3 PUFA in the sn-1 position eluted faster than the enantiomer with the n-3 PUFA in the sn-3 position, regardless of the carbon chain length and number of double bonds of the PUFA. TAG enantiomers containing DHA exhibited highly different retention on the chiral column and were separated after the first column, whereas recycling was needed to separate the enantiomer pairs containing ALA or EPA. The system using two identical columns and one mobile phase, without sample derivatization, proved to be very effective also for peak purity assessment, confirming the enantiopurity of the synthesized structured TAGs being higher than 98 % (96 % ee).

Trace water activity could improve the formation of 1,3-oleic-2-medium chain-rich triacylglycerols by promoting acyl migration in the lipase RM IM catalyzed interesterification.

New structured lipids with 1,3-oleic-2-medium chain (OMO) triacylglycerols were synthesized by promoting acyl migration in Lipozyme RM IM catalyzed interesterification between coconut oil (CO) and high oleic rapeseed oil (HORO). Results from an orthogonal design L25(55) showed that the maximal yield of OMO-structured triacylglycerols was 45.65% under the following conditions: the molar ratio of CO to HORO, 50:50; enzyme dosage, 12 wt%; reaction temperature, 60 °C; reaction time, 2 h; water activity, 0.07. Low water activity showed a high rate of acyl migration (10.86% vs 5.07% no water system), which promoted OMO synthesis due to medium-chain fatty acid migration to the sn-2 position. In a low water content (5%) system of the molecular dynamics simulation, water molecules stabilized the whole structure of RM IM through hydrogen bonding, which helped fix lipase-catalyzed active sites, making substrates more easily inserted into active sites, resulting in increased enzyme activity.

Preparation of Structured Lipid Enriched with Medium Chain Triacylglycerol by Chemical Catalyzed Acidolysis of Coconut Oil: Optimized by Response Surface Methodology.

Medium chain triacylglycerols (MCTs) have gained wide attention due to its ability to induce the residual glyceride lipolysis and improve fat absorption. In this study, structured lipid enriched with MCTs was synthesized by chemical catalyzed acidolysis of coconut oil with the mixture of caprylic acid (Cy) and capric acid (Ca) in a solvent-free system. Three catalysts were compared for their efficiency in the production of MCTs yield. The results indicated that the highest yield of MCTs was achieved by H2SO4. Effects of reaction variables on the acidolysis reaction were optimized using response surface methodology, and the optimum conditions were as follows: molar ratio of Cy to Ca 1:1, reaction time 4 h, molar ratio of MCFAs to coconut oil 12:1, catalyst loading 12 wt%, reaction temperature 110℃. Under these conditions, the obtained structured lipid contained 89.5% of MCFAs and at least 82.0% of MCTs. This paper provides a simple and low-cost method for preparing structured lipid enriched with MCTs.

Preparation of High-Purity Trilinolein and Triolein by Enzymatic Esterification Reaction Combined with Column Chromatography.

High-purity trilinolein and triolein were prepared by Novozym 435-catalyzed esterification reaction combined with column chromatography purification in this study. Firstly, linoleic acid and oleic acid were respectively extracted from safflower seed oil and camellia seed oil by urea adduct method. Secondly, trilinolein and triolein were synthesized through Novozym 435 catalyzed esterification of glycerol and fatty acids. The best synthesis conditions were obtained as follows: reaction temperature 100°C, residual pressure 0.9 kPa, enzyme dosage 6%, molar ratio of glycerol to linoleic acid 1:3 and reaction time 8 h. Crude trilinolein and triolein were further purified by silica gel column chromatography. Finally, highpurity trilinolein (95.43±0.97%) and triolein (93.07±1.05%) were obtained.

Facile lipase-catalyzed synthesis of a chocolate fat mimetic.

A cocoa butter equivalent (CBE) was synthesized enzymatically from readily available edible fats with fatty acid and triacylglycerol compositions that closely resemble the fat present in chocolate, cocoa butter. A commercially available immobilized fungal lipase, Lipozyme RM IM, was used as the reaction catalyst. Reaction parameters were a temperature of 65 °C, water activity of 0.11, a 4 h reaction time, and a substrate mass ratio of a commercial enzymatically synthesized shea stearin (SS) to palm mid-fraction (PMF) of 6:4 (w/w). Fractionation was also used after reaction completion to further approach the triacylglycerol composition of cocoa butter by removing trisaturated and unsaturated triacylglycerols. The yield of the triglyceride 1-palmitoyl-2-oleoyl, 3-stearoyl-glycerol (POS) produced was 57.7% (w/w). The amounts of 1,3-dipalmitoyl-2-oleoyl-glycerol (POP), (POS) and 1,3-stearoyl-2-oleoyl-glycerol (SOS) in the final CBE were 11.2%, 36.3%, and 34.8%, respectively. In comparison, the amounts of POP, POS and SOS in the cocoa butter used in this study were 15.2%, 38.2%, and 27.8%, respectively. No significant differences (P > 0.05) in melting point and enthalpy of fusion between CB and the CBE were observed. In comparison, a non-interesterified blend of SS and PMF (60:40 w/w) showed significantly (P < 0.05) higher melting point and lower enthalpy of fusion compared to CB. The crystal polymorphic form V of CB (ß2-3L) was similar to that of CBE and SS/PMF (60:40 w/w). The solid fat content (SFC) vs. temperature profile of the CBE generally resembled that of CB, except that the CBE had significantly (P < 0.05) higher SFCs at 5, 10, 15, 20 and 25 °C compared to both CB and SS/PMF (60:40 w/w). Addition of 15% (w/w) CBE to CB did not cause any changes in physical properties (melting point, SFC and crystal polymorphic forms) of the CB. This study demonstrates the potential for synthesizing a CB-like CBE using a green, rapid, straightforward one step enzymatic conversion followed by fractionation from widely available edible fats.

Cyclopropane fatty acid biosynthesis in plants: phylogenetic and biochemical analysis of Litchi Kennedy pathway and acyl editing cycle genes.

KEY MESSAGE: This report describes the most extensive known gene discovery study from an oilseed that produces cyclopropane fatty acids, a novel industrial feedstock. Nature contains hundreds of examples of plant species that accumulate unusual fatty acids in seed triacylglycerols (TAG). Although lipid metabolic genes have been cloned from several exotic plant species, the underlying mechanisms that control the production of novel TAG species are still poorly understood. One such class of unusual fatty acids contain in-chain cyclopropane or cyclopropene functionalities that confer chemical and physical properties useful in the synthesis of lubricants, cosmetics, dyes, coatings, and other types of valuable industrial feedstocks. These cyclopropyl fatty acids, or CPFAs, are only produced by a small number of plants, primarily in the order Malvidae. Litchi chinensis is one member of this group; its seed oil contains at least 40 mol% CPFAs. Several genes, representing early, middle, and late steps in the Litchi fatty acid and TAG biosynthetic pathways have been cloned and characterized here. The tissue-specific and developmental transcript expression profiles and biochemical characteristics observed indicate which enzymes might play a larger role in Litchi seed TAG biosynthesis and accumulation. These data, therefore, provide insights into which genes likely represent the best targets for either silencing or overexpression, in future metabolic engineering strategies aimed at altering CPFA content.

Intensified synthesis of medium chain triglycerides using ultrasonic reactors at a capacity of 4L.

Lipids are considered as one of the most crucial nutrients for humans and among the various classes, medium chain triglycerides (MCTs) are considered as the most important functional foods and nutraceuticals. The present work deals with the intensification of synthesis of MCTs at a large capacity of 4L based on the use of ultrasonic bath and ultrasonic longitudinal horn. The effect of operating parameters like molar ratio of the reactants, type of catalyst and catalyst loading as well as the temperature on the extent of conversion has been investigated. The effect of molar ratio of lauric acid and glycerol was investigated over the range of 1:2 to 1:8 whereas the effect of loading of sulfuric acid was studied over the range of 4 ml/L-10 ml/L and zinc chloride loading over the range of 1 g/L-4 g/L. The effect of temperature was also studied using the conventional approach where it has been observed that 90 °C is an optimum temperature giving the extent of conversion as 72%. Also, the use of homogeneous catalyst as sulphuric acid was found to be more effective as compared to the solid catalyst as zinc chloride. It was observed that the maximum extent of conversion as 77.5% was obtained at 8 ml/L of sulfuric acid and molar ratio of 1:6 using ultrasonic longitudinal horn with US bath giving lower conversion as compared to US longitudinal horn but higher than the conventional approach under same operating conditions. The present work clearly established the intensification benefits in terms of reduction in time and higher conversion using cavitational reactors.

Preparation of Chiral Triacylglycerols, sn-POO and sn-OOP, via Lipase-mediated Acidolysis Reaction.

It is well known that lipases are useful tools for preparing various structured triacylglycerols (TAGs). However, the lipase-mediated preparation of chiral TAGs has never been reported. This study aimed to prepare chiral TAGs (viz., 1-palmitoyl-2,3-dioleoyl-sn-glycerol (sn-POO) or 1,2-dioleoyl-3-palmitoyl-sn-glycerol (sn-OOP)) via lipase mediated acidolysis, using triolein (TO) and palmitic acid (P) as substrates. Three commercially available lipases (viz., Lipozyme RM-IM®, Lipozyme TL-IM®, and Lipase OF®) were used. Lipozyme RM-IM® resulted in an increase 1P-2O (sn-POO + sn-OOP + 1,3-dioleoyl-2-palmitoyl-sn-glycerol) content with reaction time, which plateaued at 2~24 h (max. yield 47.1% at 4 h). The highest sn-POO/sn-OOP ratio of ca. 9 was obtained at 0.25 h, and the rate got close to 1 with reaction time (sn-POO/sn-OOP = 1.3 at 24 h). Lipozyme TL-IM® resulted in a lower 1P-2O synthesis rate than Lipozyme RM-IM®, where its highest sn-POO/sn-OOP ratio of ca. 2 was obtained at 0.25 h and did not vary much further with reaction time. In the case of Lipase OF®, its reaction rate for 1P-2O synthesis was lower than that of the other two lipases, and the highest sn-POO/sn-OOP ratio of ca. 1.4 was obtained at 0.5 h, reaching closer to 1 with a longer reaction time. Reaction solvents (viz., hexane, acetone, and benzene) also affected the 1P-2O preparation, where the highest 1P-2O content was obtained with the solvent-free system. Furthermore, the solvent-free system showed a higher reaction rate for 1P-2O synthesis than did the hexane system, with no effect on chiral specificity of the lipase for the TAG molecules. These results suggested that among three types of commercial lipase, Lipozyme RM-IM® is the most useful for the preparation of chiral TAGs by acidolysis reaction.

Mannose-functionalized solid lipid nanoparticles are effective in targeting alveolar macrophages.

Mannose receptor is highly expressed on alveolar macrophages, being a potential target to promote the specific local drug delivery of anti-tuberculosis agents through the use of functionalized nanocarriers. In this work, isoniazid (Isn)-loaded solid lipid nanoparticles (SLN), reinforced with stearylamine (SA) were produced by double emulsion technique and further surface-functionalized with mannose in a straightforward chemical approach. Upon pre-formulation assessment, SLN close to 500 nm average size, positively charged and with association efficiency of ISN close to 50% were obtained. Functionalization with mannose was performed after SLN production and confirmed by Fourier transform infrared spectroscopy (FTIR). Both functionalized and non-functionalized SLN demonstrated to devoid of toxicity when tested in human lung epithelial cell line (NCI-H441) and differentiated THP-1 (dTHP-1), reducing the intrinsic cytotoxicity of Isn when incorporated into SLN. Uptake studies were conducted on same macrophage-like cells and the results showed that fluorescent mannosylated SLN (M-SLN) were more efficient in be internalized comparatively to SLN. Moreover, the uptake of M-SLN was reduced when cells were pre-incubated with mannose, demonstrating the receptor-dependence internalization of functionalized SLN. These functionalized nanocarriers may represent a useful platform to target alveolar macrophages for delivering anti-infective drugs.

Synthesis of 1,3-distearoyl-2-oleoylglycerol by enzymatic acidolysis in a solvent-free system.

1,3-Distearoyl-2-oleoylglycerol (SOS) is widely used as a cocoa butter improver and an anti-blooming agent in the chocolate industry. In this study, an effective process was developed to prepare SOS by enzymatic acidolysis. Under optimal reaction conditions (substrate molar ratio of 12, NS40086 loading of 10%, a solvent-free system, 75°C for 4h), a SOS yield of 70.2% was obtained. Subsequently, two-step purification was applied to purify the crude SOS product. Free fatty acids were completely removed after molecular distillation. After acetone fractionation, the purity of SOS reached 92.2% with an 85.1% recovery. Comparison of various physicochemical properties of purified SOS and a commercial cocoa butter improver showed their properties to be similar. This study provides an effective and sustainable process for the synthesis of SOS product, which is expected to be used as a high-quality cocoa butter improver or an anti-blooming agent in the chocolate industry.

Facile preparation of magnetic carbon nanotubes-immobilized lipase for highly efficient synthesis of 1,3-dioleoyl-2-palmitoylglycerol-rich human milk fat substitutes.

In this study, Candida lipolytica lipase (CLL) was immobilized on magnetic multi-walled carbon nanotubes (mMWCNTs) via hydrophobic and cation-exchange interaction. The resultant immobilized CLL showed much better thermal stability, biocatalyst activity and easier recycling than the free form. A method for efficient enzymatic acidolysis of tripalmitin (PPP) with oleic acid (OA), to produce OPO-rich TAGs, was developed, using the immobilized CLL as the biocatalyst. Under optimized conditions (2% water, 20mg/ml enzyme, 1:6 PPP/OA, 50°C, 2h), the content of OPO in the final product reached 46.5%. CLL@mMWCNTs had a better activity and manipulative stability than commercial lipases. More importantly, the feasibility of CLL@mMWCNTs was also validated in the practical production of OPO-rich TAGs, using lard and restructured palm oil as the raw material. These results suggest that CLL@mMWCNTs is a promising biocatalyst for the OPO-rich TAGs production and will be helpful for the infant formula industry.

Synthesis and evaluation of fluorogenic triglycerides as lipase assay substrates.

Three racemic fluorogenic triglycerides are synthesized and evaluated as lipase assay substrates. The presented synthesis route goes through a key triglyceride intermediate which can be chemoselectively functionalized with a wide range of different probes. Hence the substrate can be tailor-made for a specific assay, or focus can be on low cost in larger scale for applications in high-throughput screening (HTS) assays. In the specific examples, TG-ED, TG-FD and TG-F2 are assembled with the Edans-Dabcyl or the fluorescein-Dabcyl FRET pair, or relying on fluorescein self-quenching, respectively. Proof-of-concept assays allowed determination of 1st order kinetic parameters (kcat/KM) of 460s(-1)M(-1), 59s(-1)M(-1) and 346s(-1)M(-1), respectively, for the three substrates. Commercially available EnzChek lipase substrate provided 204s(-1)M(-1). Substrate concentration was identified as a critical parameter, with measured reaction rates decreasing at higher concentrations when intermolecular quenching becomes significant.

An insight on acyl migration in solvent-free ethanolysis of model triglycerides using Novozym 435.

In this work, the ethanolysis of triglycerides catalyzed by immobilized lipase was studied, focusing on the secondary reaction of acyl migration. The catalytic tests were performed in a solvent-free reaction medium using Novozym 435 as biocatalyst. The selected experimental variables were biocatalyst loading (5-20mg), reaction time (30-90min), and chain length of the fatty acids in triglycerides with and without unsaturation (short (triacetin), medium (tricaprylin) and long (tripalmitin/triolein)). The formation of 2-monoglyceride by ethanolysis of triglycerides was favored by long reaction times and large biocatalyst loading with saturated short- to medium-chain triglycerides. In the case of long-chain triglycerides, the formation of this monoglyceride was widely limited by acyl migration. In turn, acyl migration increased the yield of ethyl esters and minimized the content of monoglycerides and diglycerides. Thus, the enzymatic synthesis of biodiesel was favored by long-chain triglycerides (which favor the acyl migration), long reaction times and large biocatalyst loading. The conversion of acylglycerides made from long-chain fatty acids with unsaturation was relatively low due to limitations in their access to the active site of the lipase.

Production of Structured Triacylglycerols Containing Palmitic Acids at sn-2 Position and Docosahexaenoic Acids at sn-1, 3 Positions.

Docosahexaenoic acid supplementation has been shown well-established health benefits that justify their use as functional ingredients in healthy foods and nutraceutical products. Structured triacylglycerols rich in 1,3-docosahexenoyl-2-palmitoyl-sn-glycerol were produced from algal oil (Schizochytrium sp) which was prepared by a two-step process. Novozym 435 lipase was used to produce tripalmitin. Tripalmitin was then used to produce the final structured triacylglycerol (STAG) through interesterification reactions using Lipozyme RM IM. The optimum conditions for the enzymatic reaction were a mole ratio of tripalmitin/fatty acid ethyl esters 1:9, 60°C, 10% enzyme load (wt % of substrates), 10 h; the enzymatic product contained 51.6% palmitic acid (PA), 30.13% docosahexaenoic acid (DHA, C22:6 n-3) and 5.33% docosapentanoic acid (DPA, C22:5 n-3), 12.15% oleic acid (OLA). This STAG can be used as a functional ingredient in dietary supplementation to provide the benefits of DHA.

Osage Orange (Maclura pomifera L.) Seed Oil Poly(α-hydroxydibutylamine) Triglycerides: Synthesis and Characterization.

Milled Osage orange seeds (Maclura pomifera (Raf.) Schneid) were Soxhlet extracted with hexane, and portions of the extract were treated with activated carbon before solvent removal. The crude oil was winterized and degummed by centrifugation at low temperature. Decantation of the centrifugate gave an admixture of the triglycerides and free fatty acids. The free fatty acid content of the oil was removed when portions of the admixture were diluted with hexane and shaken with cold aqueous ammonium hydroxide (0.1 M) solution. The desiccant-dried organic phase was concentrated under reduced pressure to give the cleaned Osage orange triglyceride after solvent removal by rotary evaporation at 67 °C. Epoxidation of the resulting cleaned triglyceride was effected by reaction with in situ generated peroxy performic acid in H2O2. The oxirane rings of the derivatized oil were then opened using N,N-dibutylamine catalyzed by anhydrous ZnCl2 to afford the poly(α-hydroxydibutylamine) triglyceride. The purpose of this work was to derivatize and thereby stabilize this highly unsaturated tree oil for its eventual use in lubrication applications.

Polyamine Triglycerides: Synthesis and Study of Their Potential in Lubrication, Neutralization, and Sequestration.

Renewable resources have evoked a new awakening in both scientific and industrial circles in the past decade. Vegetable oil is one category of renewables that is amenable as a source of new industrial products. Because the source feedstock, seeds, are environmentally friendly, the derivatized products from these at the end of their lifetime could also be benign when designed appropriately. Bioethanol and biodiesel are examples of biobased industrial products currently in the market place and have become resources for uplifting the rural economy. Biolubricants also are playing a more prominent role because they have become closely competitive with petroleum-based lubricants. These products are renewable because the crops from which the feedstuff for the biofuels and biolubricants are produced are grown annually in contrast to nonrenewable mineral sources. Added to their renewability is the inherent biodegradability of their end-use products after their useful lifetime. In a recent study of the lubricity characteristics of peracylated polyhydroxy milkweed oil, the derivatives were found to exhibit good oxidative stability as well as excellent antiwear properties. To further explore an expansion in the properties of such materials in lubrication and other applications, in this study the polyhydroxy (OH) moieties of derivatized milkweed triglycerides were replaced with -NHR groupings in the oil. In this process novel polyketo triglyceride intermediates leading to polyamine derivatives of the vegetable oil have been synthesized. The polyamine triglyceride markedly improved the stability of the parent oil to oxidative stress. It has also attenuated the extreme viscosity of the starting polyhydroxy oil to a more useful product that could be amenable for use as a lubricating agent, for example, hydraulic fluid. Both the polyketone and polyimine intermediates of the polyamine have chelating properties. The intermediates and the polyamine were characterized spectroscopically, tribologically, and rheologically for their intrinsic properties.

Enzymatic synthesis of triacylglycerols of docosahexaenoic acid: Transesterification of its ethyl esters with glycerol.

The synthesis of docosahexaenoyl triacylglycerides at low temperature (e.g., 50°C) using biocatalysts of 6 commercial lipases adsorbed on hydrophobic supports was studied. In general, the triacylglyceride yields were very low with the exceptions of those produced with the enzymes from Candida antarctica fraction B, CALB (82%), and those produced with the enzyme from Pseudomonas fluorescens, PFL (57%). The reactions were performed under vacuum to remove the released ethanol. The yields varied widely when different derivatives of CALB were used, and they were higher when CALB adsorbed on hydrophobic supports was used (82%). One interesting by-product (18% of sn-2 monoacylglyceride of DHA) remained at the end of the synthetic process. CALB adsorbed on Sepabeads exhibited better activity and stability than did the commercial derivative Novozym 435. The best CALB biocatalyst preserved 90% of the activity after 30days under the reaction conditions.

Ultrasonic pretreatment in lipase-catalyzed synthesis of structured lipids with high 1,3-dioleoyl-2-palmitoylglycerol content.

Production of structured lipid 1,3-dioleoyl-2-palmitoylglycerol (OPO), from tripalmitin (PPP) and oleic acid (OA) using lipases and ultrasonic pretreatment was conducted. Factors influencing both the ultrasonic conditions and enzymatic reaction were investigated. Optimum conditions could be attained with 6 min pretreatment time, 50% ultrasonic power, 3 s/9 s (work/pause) cycle of ultrasonic pulse, 1:8 PPP/OA molar ratio, 12% enzyme dosage and 50 °C temperature of. At the optimum conditions, the OPO yield of 51.8% could be achieved in 4h. Studies showed that the OPO content increased to 35.9% in 1h with ultrasonic pretreatment, in comparison to 4h without ultrasonic pretreatment. Reuse of Lipozyme RM IM for 10 cycles under ultrasonic irradiation did not cause essential damage to its lipase activity. Reaction kinetic model fitted well with the proposed Ping-Pong mechanism. The apparent kinetic constant (Vm'/K2) of ultrasound pretreatment reaction was 2.52 times higher than the conventional mechanical stirring, indicating that ultrasound pretreatment enhanced the substrates affinity to the enzyme. This study confirmed that ultrasonic pretreatment was more efficient in OPO production than conventional mechanical agitation.