Diacylglycerol Production Research Articles

Diacylglycerol production by genetically modified lipase from Malassezia globosa

Diacylglycerol (DAG)-enriched oil has drawn considerable attention for the prevention of obesity and other lifestyle-related diseases. In this study, a mutant lipase of SMG1 (SMG1-F278D) from Malassezia globosa was studied for the production of DAG. The SMG1-F278D exhibited 4-fold increased esterification activity as well as superior fatty acid (FA) specificity for medium chain FAs. Molecular docking study suggested that the caprylic acid (CA) was strongly bound to the catalytic residue Ser171 present in the SMG1-F278D as compared to SMG1. Molecular Dynamics (MD) simulations were employed in order to understand the structural conformations of SMG1 and SMG1-F278D. The structure of SMG1-F278D was found to be more compact as well as less deviated from its native conformation. There was an increase in β-sheet as well as α-helix in the SMG1-F278D that may stabilize the protein structure due to point mutation. Finally, the capability of SMG1-F278D in synthesis of DAG was evaluated. Effects of reaction parameters such as substrate molar ratio of glycerol to FAs, enzyme loading and reaction temperature on the esterification were investigated. The optimal reaction conditions were achieved as 4:1 molar ratio of glycerol to FAs, enzyme loading of 100U/g (U/w, with respect to the total substrates) and temperature of 25°C. The present study provides important information about SMG1 mutant for better utilization in the oils and fats industries.

Phosphatidic Acid Sequesters Sec18p from cis-SNARE Complexes to Inhibit Priming.

Yeast vacuole fusion requires the activation of cis-SNARE complexes through priming carried out by Sec18p/N-ethylmaleimide sensitive factor and Sec17p/α-SNAP. The association of Sec18p with vacuolar cis-SNAREs is regulated in part by phosphatidic acid (PA) phosphatase production of diacylglycerol (DAG). Inhibition of PA phosphatase activity blocks the transfer of membrane-associated Sec18p to SNAREs. Thus, we hypothesized that Sec18p associates with PA-rich membrane microdomains before transferring to cis-SNARE complexes upon PA phosphatase activity. Here, we examined the direct binding of Sec18p to liposomes containing PA or DAG. We found that Sec18p preferentially bound to liposomes containing PA compared with those containing DAG by approximately fivefold. Additionally, using a specific PA-binding domain blocked Sec18p binding to PA-liposomes and displaced endogenous Sec18p from isolated vacuoles. Moreover, the direct addition of excess PA blocked the priming activity of isolated vacuoles in a manner similar to chemically inhibiting PA phosphatase activity. These data suggest that the conversion of PA to DAG facilitates the recruitment of Sec18p to cis-SNAREs. Purified vacuoles from yeast lacking the PA phosphatase Pah1p showed reduced Sec18p association with cis-SNAREs and complementation with plasmid-encoded PAH1 or recombinant Pah1p restored the interaction. Taken together, this demonstrates that regulating PA concentrations by Pah1p activity controls SNARE priming by Sec18p.

Modeling and Optimization of Lipase-Catalyzed Partial Hydrolysis for Diacylglycerol Production in Packed Bed Reactor

Abstract Response surface methodology (RSM) was employed to optimize the process variables namely packed bed height (cm) and flow rates (ml/min) on diacylglycerol (DAG) production via partial hydrolysis of palm oil using immobilized Rhizomucor miehei lipase in packed bed reactor (PBR). Quadratic models were successfully developed for both DAG(y) and unhydrolyzed triacylglycerol ((un)TAG) with determination coefficient (R 2) of 0.9931 and 0.9986, respectively coupled with insignificant lack of fit (p > 0.05). Optimal conditions for DAG synthesis were evaluated to be 10 cm packed bed height and 3.8 ml/min flow rate. Immobilized enzyme can be reused up to 10 times without significant changes in enzymatic activity. The partial hydrolysis under studied was found to be mass transfer-controlled.

Age-related changes in retinoic, docosahexaenoic and arachidonic acid modulation in nuclear lipid metabolism

The aim of this work was to study how age-related changes could modify several enzymatic activities that regulate lipid mediator levels in nuclei from rat cerebellum and how these changes are modulated by all-trans retinoic acid (RA), docosahexaenoic acid (DHA) and arachidonic acid (AA). The higher phosphatidate phosphohydrolase activity and lower diacylglycerol lipase (DAGL) activity observed in aged animals compared with adults could augment diacylglycerol (DAG) availability in the former. Additionally, monoacylglycerol (MAG) availability could be high due to an increase in lysophosphatidate phosphohydrolase (LPAPase) activity and a decrease in monocylglycerol lipase activity. Interestingly, RA, DHA and AA were observed to modulate these enzymatic activities and this modulation was found to change in aged rats. In adult nuclei, whereas RA led to high DAG and MAG production through inhibition of their hydrolytic enzymes, DHA and AA promoted high MAG production by LPAPase and DAGL stimulation. In contrast, in aged nuclei RA caused high MAG generation whereas DHA and AA diminished it through LPAPase activity modulation. These results demonstrate that aging promotes a different nuclear lipid metabolism as well as a different type of non-genomic regulation by RA, DHA and AA, which could be involved in nuclear signaling events.

Phosphorylation of Yeast Pah1 Phosphatidate Phosphatase by Casein Kinase II Regulates Its Function in Lipid Metabolism

Pah1 phosphatidate phosphatase in Saccharomyces cerevisiae catalyzes the penultimate step in the synthesis of triacylglycerol (i.e. the production of diacylglycerol by dephosphorylation of phosphatidate). The enzyme playing a major role in lipid metabolism is subject to phosphorylation (e.g. by Pho85-Pho80, Cdc28-cyclin B, and protein kinases A and C) and dephosphorylation (e.g. by Nem1-Spo7) that regulate its cellular location, catalytic activity, and stability/degradation. In this work, we show that Pah1 is a substrate for casein kinase II (CKII); its phosphorylation was time- and dose-dependent and was dependent on the concentrations of Pah1 (Km = 0.23 μm) and ATP (Km = 5.5 μm). By mass spectrometry, truncation analysis, site-directed mutagenesis, phosphopeptide mapping, and phosphoamino acid analysis, we identified that >90% of its phosphorylation occurs on Thr-170, Ser-250, Ser-313, Ser-705, Ser-814, and Ser-818. The CKII-phosphorylated Pah1 was a substrate for the Nem1-Spo7 protein phosphatase and was degraded by the 20S proteasome. The prephosphorylation of Pah1 by protein kinase A or protein kinase C reduced its subsequent phosphorylation by CKII. The prephosphorylation of Pah1 by CKII reduced its subsequent phosphorylation by protein kinase A but not by protein kinase C. The expression of Pah1 with combined mutations of S705D and 7A, which mimic its phosphorylation by CKII and lack of phosphorylation by Pho85-Pho80, caused an increase in triacylglycerol content and lipid droplet number in cells expressing the Nem1-Spo7 phosphatase complex.

Secreted lipases from Malassezia globosa: recombinant expression and determination of their substrate specificities.

Malassezia globosa, which is associated with skin conditions such as dandruff and seborrhoeic dermatitis, possesses 13 secreted lipases, but only MgLip1, MgMDL2 and MgLip2 have been characterized. To understand the substrate preferences of these lipases and by extension their potential role in colonizing human skin, we expressed all 13 predicted secreted lipases in Pichia pastoris and evaluated their ability to utilize mono-, di- and triolein substrates. The M. globosa family class 3 lipases were shown to be specific for mono- and diacylglycerols, but exhibited no regio-selective production of diacylglycerols, which are of special interest for industrial applications. Lipases belonging to the Lip family utilized all substrates. In a further step, five lipases previously demonstrated to be expressed on human skin were tested against the eight most common di- and triacylglycerols in human sebum. All lipases liberated free fatty acids from three to eight of these substrates, proving their ability to hydrolyse key components of human sebum. Again, only Lip family lipases showed activity on triacylglycerides. Based on the demonstrated activity and expression levels of MgLip2 in M. globosa, the Lip lipase family appears to have the highest impact for the pathogenicity of M. globosa.

Fast Production of Diacylglycerol in a Solvent Free System via Lipase Catalyzed Esterification Using a Bubble Column Reactor

AbstractIn this study, diacylglycerols (DAG) were synthesized rapidly (~30 min) in a solvent‐free system via esterification of glycerol with fatty acids (FA, the mixture of 60 wt% palm oil deodorizer distillate and 40 wt% oleic acid) catalyzed by Lipozyme 435 (Novozymes A/S, Copenhagen, Denmark) using a bubble column reactor. The content of DAG, monoacylglycerols (MAG), triacylglycerols (TAG) and free fatty acids (FFA) in the crude product were 57.94 ± 1.60 wt%, 24.68 ± 2.08 wt%, 2.67 ± 1.72 wt% and 14.69 ± 1.22 wt%, respectively under the selected conditions, which were enzyme load of 5.0 wt%, glycerol/FA mole ratio of 7.5, initial water content of 2.5 wt%, reaction temperature of 60 °C, reaction time of 30 min and N2 gas flow of 10.6 cm min−1. The final product containing 91.30 ± 1.10 wt% of DAG was obtained by one‐step molecular distillation at 200 °C. The reusability of Lipozyme 435 was investigated by evaluating the esterification degree (ED) and the DAG content in the crude products in 30 successive runs. The enzyme retained 95.10 % of its original activity during 30 successive runs according to comparison of the ED. The new process showed a very high efficiency in production of DAG with a high purity. The ratio of positional isomers 1,3‐DAG to 1,2 ‐DAG was 2:1 in the final product. The certain plasticity (melting point of 44 °C) and content of unsaturated fatty acids made the product a valuable food ingredient.

Measurement of Inositol Triphosphate Levels from Rat Hippocampal Slices.

Inositol triphosphate (IP3) is an important second messenger that participates in signal transduction pathways in diverse cell types including hippocampal neurons. Stimulation of phospholipase C in response to various stimuli (hormones, growth factors, neurotransmitters, neurotrophins, neuromodulators, odorants, light, etc) results in hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, and leads to the production of IP3 and diacylglycerol. Binding of IP3 to the IP3 receptor (IP3R) induces Ca2+ release from intracellular stores and enables the initiation of intracellular Ca2+-dependent signaling. Here we describe a procedure for the measurement of cellular IP3 levels in tissue homogenates prepared from rat hippocampal slices.

Diabetes prevalence in NZO females depends on estrogen action on liver fat content.

In humans and rodents, risk of metabolic syndrome is sexually dimorphic, with an increased incidence in males. Additionally, the protective role of female gonadal hormones is ostensible, as prevalence of type 2 diabetes mellitus (T2DM) increases after menopause. Here, we investigated the influence of estrogen (E2) on the onset of T2DM in female New Zealand obese (NZO) mice. Diabetes prevalence (defined as blood glucose levels >16.6 mmol/l) of NZO females on high-fat diet (60 kcal% fat) in week 22 was 43%. This was markedly dependent on liver fat content in week 10, as detected by computed tomography. Only mice with a liver fat content >9% in week 10 plus glucose levels >10 mmol/l in week 9 developed hyperglycemia by week 22. In addition, at 11 wk, diacylglycerols were elevated in livers of diabetes-prone mice compared with controls. Hepatic expression profiles obtained from diabetes-prone and -resistant mice at 11 wk revealed increased abundance of two transcripts in diabetes-prone mice: Mogat1, which catalyzes the synthesis of diacylglycerols from monoacylglycerol and fatty acyl-CoA, and the fatty acid transporter Cd36. E2 treatment of diabetes-prone mice for 10 wk prevented any further increase in liver fat content and reduced diacylglycerols and the abundance of Mogat1 and Cd36, leading to a reduction of diabetes prevalence and an improved glucose tolerance compared with untreated mice. Our data indicate that early elevation of hepatic Cd36 and Mogat1 associates with increased production and accumulation of triglycerides and diacylglycerols, presumably resulting in reduced hepatic insulin sensitivity and leading to later onset of T2DM.

Optimization of diacylglycerol production by glycerolysis of fish oil catalyzed by Lipozyme TL IM with Tween 65.

The diacylglycerols (DAG) are emulsifiers with high added value used as functional additives in food, medicine, and cosmetic industries. In glycerolysis of oils for the production of DAG, the immiscibility between the substrates (glycerol and oil phases) has to be overcome, for example, by the addition of a food grade surfactant like Tween 65. The main objective of this work was to optimize the process conditions for obtaining diacylglycerols rich in the omega-3 eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, through the enzymatic glycerolysis of fish oil, in systems with Tween 65 and without this surfactant, using Lipozyme TL(®) IM as biocatalyst. The experiments were performed according to predetermined conditions varying the temperature, enzyme load, the oil to glycerol molar ratio and, when added, the surfactant load. After 6 h of reaction, it was possible to produce up to 20.76 and 13.76% of diacylglycerols from fish oil in the reactions with and without Tween 65, respectively.

Asymmetrical diacylglycerol dynamics on the cytosolic and lumenal sides of a single endomembrane in living cells.

The elucidation of lipid dynamics on the cytosolic and lumenal sides of a single endomembrane has been challenging in living cells because of the lack of appropriate methods. Diacylglycerol (DAG) is a lipid second messenger that is produced by enzymes that reside on both the cytosolic and lumenal sides of the endomembrane. In the present study, we attempted to observe both the cytosolic and lumenal DAG dynamics at endomembranes including the Golgi apparatus and the endoplasmic reticulum in Madin-Darby canine kidney (MDCK) cells. We developed a Förster resonance energy transfer (FRET)–based probe to detect DAG at the luminal side (lumenal DAG) of endomembranes. In combination with the FRET-based cytosolic DAG probe that has already been established, it was found that lumenal DAG is generated in a calcium-dependent manner by thapsigargin, which increases cytosolic calcium concentrations. In contrast, DAG production at the cytosolic side of endomembranes did not occur under the same experimental conditions. The thapsigargin-induced DAG generation was abolished by treatment with an inhibitor of sphingomyelin synthase (SMS) and phosphatidylcholine-specific phospholipase C (PC-PLC), which produce lumenal DAG. Thus, we have established a successful method for monitoring both cytosolic and lumenal DAG dynamics at the endomembrane in living cells.

Abstract 16: Tribbles1 Regulates Hepatic Lipogenesis in Mice through Post-Translational Regulation of C/EBPα

Tribbles1 (TRIB1) was recently identified in genome-wide association studies as being associated with plasma levels of VLDL, HDL, LDL, and TG as well as coronary artery disease in humans. Here we report studies in a Trib1 liver-specific knockout mouse (Trib1_LSKO) created via AAV-mediated delivery of Cre recombinase into mice with a floxed allele of Trib1. Four weeks after infection, Trib1_LSKO mice exhibit a 25% increase in liver weight (p<0.01) as compared to controls given null virus, and examination of hepatic lipids reveals a 78% increase in TG content (p<0.001) of Trib1_LSKO livers. Hepatic transcription of lipogenic genes (i.e. Scd1, Fasn, and Acaca) was increased greater than 2-fold in Trib1_LSKO mice, and when in vivo lipogenesis was measured, Trib1_LSKO animals exhibited significantly increased hepatic production of fatty acids (2.2-fold, p=0.02), diacylglycerol (1.8-fold, p<0.01), and TG (3.6-fold, p<0.001). Microarray and subsequent pathway analysis of Trib1_LSKO livers revealed altered transcription of genes downstream of the transcription factor C/EBPα, and western blot analysis of liver extracts showed increased protein levels of C/EBPα in Trib1_LSKO mice compared to controls. C57Bl/6 mice overexpressing C/EBPα via AAV (AAV_Cebpa) exhibit increases in hepatic production of TG (3.6-fold, p<0.001), fatty acids (2.5-fold, p<0.001), and diacylglycerol (2.1-fold, p<0.005) that mirror those observed in Trib1_LSKO mice. Interestingly, while Trib1_LSKO mice have altered plasma lipids, AAV_Cebpa animals do not, suggesting that Trib1 modulates plasma lipids via a mechanism separate from C/EBPα-mediated lipogenesis. Finally, ChIP-Seq analysis of C/EBPα DNA occupancy in Trib1_LSKO and AAV_Cebpa mice showed increased binding near lipogenic genes as well as upstream of Trib1 itself. Trib1 does show increased gene expression in response to C/EBPα overexpression, suggesting that Trib1 and C/EBPα participate in a feedback loop. In summary, we demonstrate here that Trib1 regulates de novo lipogenesis through modulation of hepatic protein levels of C/EBPα, which in turn regulates Trib1 expression. These data assign novel roles for both Trib1 and C/EBPα in the regulation of hepatic lipid metabolism.

DIACYLGLYCEROL ACCUMULATION IMPAIRS SHORT-TERM ACTIVATION OF PHOSPHOLIPASE D BY THYROXINE IN THE LIVER CELLS

Thyroid hormones (TG) are known modulators of signal transduction. Phospholipase D (PLD) is one of the targets of TG in the stimulated cells. Response of cells to the short-term TG action significantly reduces at old age. Taking into account that diacylglycerol (DAG) accumulation induces the resistance of cells to some of regulatory factors in the target cells the aim of the present study was to determine if DAG content increase in hepatocytes impairs the L-thyroxine (L-T4) short-term action. The experiments were performed in either the [14C]palmitic acid- labeled hepatocytes or [14C]oleic acid-pre-labeled liver cells of 3- and 24-month-old rats. To study the short-term L-T4 action on cells the PLD activation was determined. The DAG production and content in hepatocytes significantly increased at old age and in the young cells pre-treated with palmitic acid. The reduction of DAG level in cells by means of DAG-kinase activator, alfa-tocoferol acetate, or long-term L-T4 treatment improved the short-term hormone action. The above data have indicated that DAG play important role in the L-T4 PLD regulation. The cross-talk between classic and non-genomic pathways of TG regulation of lipid metabolism has been determined.

Diacylglycerol Kinase ζ (DGKζ) Is a Critical Regulator of Bone Homeostasis Via Modulation of c-Fos Levels in Osteoclasts.

Increased diacylglycerol (DAG) levels are observed in numerous pathologies, including conditions associated with bone loss. However, the effects of DAG accumulation on the skeleton have never been directly examined. Because DAG is strictly controlled by tissue-specific diacylglycerol kinases (DGKs), we sought to examine the biological consequences of DAG accumulation on bone homeostasis by genetic deletion of DGKζ, a highly expressed DGK isoform in osteoclasts (OCs). Strikingly, DGKζ(-/-) mice are osteoporotic because of a marked increase in OC numbers. In vitro, DGKζ(-/-) bone marrow macrophages (BMMs) form more numerous, larger, and highly resorptive OCs. Surprisingly, although increased DAG levels do not alter receptor activator of NF-κB (RANK)/RANK ligand (RANKL) osteoclastogenic pathway, DGKζ deficiency increases responsiveness to the proliferative and pro-survival cytokine macrophage colony-stimulating factor (M-CSF). We find that M-CSF is responsible for increased DGKζ(-/-) OC differentiation by promoting higher expression of the transcription factor c-Fos, and c-Fos knockdown in DGKζ(-/-) cultures dose-dependently reduces OC differentiation. Using a c-Fos luciferase reporter assay lacking the TRE responsive element, we also demonstrate that M-CSF induces optimal c-Fos expression through DAG production. Finally, to demonstrate the importance of the M-CSF/DGKζ/DAG axis on regulation of c-Fos during osteoclastogenesis, we turned to PLCγ2(+/-) BMMs, which have reduced DAG levels and form fewer OCs because of impaired expression of the master regulator of osteoclastogenesis NFATc1 and c-Fos. Strikingly, genetic deletion of DGKζ in PLCγ2(+/-) mice rescues OC formation and normalizes c-Fos levels without altering NFATc1 expression. To our knowledge, this is the first report implicating M-CSF/DGKζ/DAG axis as a critical regulator of bone homeostasis via its actions on OC differentiation and c-Fos expression.

Selective Production of Diacylglycerols Through Glycerolysis by Ionic Liquid: 1‐Butyl‐3‐Methylimidazolium Imidazolide as Catalyst and Reaction Medium

AbstractIn this study, 1‐butyl‐3‐methylimidazolium imidazolide ([Bmim]Im) was found effective to catalyze the glycerolysis of triacylglycerols (TAG) for selective production of diacylglycerols (DAG). About 60 % content of DAG and 80 % conversion of TAG were achieved with [Bmim]Im at 15 % (wt% based on TAG), without requirement of any other catalyst and reaction medium. In addition, the weight ratio of DAG to monoacylglycerols (MAG) was at 0.9 when the glycerol/TAG mole ratio was 5:1, indicating [Bmim]Im was selective for DAG generation.

Lipase-Catalyzed Glycerolysis of Soybean and Canola Oils in a Free Organic Solvent System Assisted by Ultrasound.

This work shows new and promising experimental data of soybean oil and canola oil glycerolysis using Novozym 435 enzyme as catalyst in a solvent-free system using ultrasound bath for the emulsifier, monoglyceride (MAG), and diacylglycerol (DAG) production. The experiments were conducted in batch mode to study the influence of process variables as temperature (40 to 70°C), immobilized enzyme content (2.5 to 10wt%, relative to substrates), molar ratio glycerol/oil (0.8:1 to 3:1), agitation (0 to 1200rpm) and ultrasound intensity (0 to 132W cm(-2)). Highest yields of DAG+MAG (75wt%) were obtained with molar ratio glycerol/canola oil 0.8:1, 70°C, 900rpm, 120min of reaction time, 10wt% of enzyme concentration, and 52.8W cm(-2) of ultrasound intensity. When soybean oil was used, the best results in terms of DAG+MAGs (65wt%) were using molar ratio of glycerol/soybean oil 0.8:1, 70°C, 900rpm, 90min of reaction time, 10wt% of enzyme content, and 40% of ultrasound intensity (52.8W cm(-2)). The results showed that the lipase-catalyzed glycerolysis in a solvent-free system with ultrasound bath can be a potential route for high content production of DAGs and MAGs.

Divergence in endothelin-1- and bradykinin-activated store-operated calcium entry in afferent sensory neurons.

Endothelin-1 (ET-1) and bradykinin (BK) are endogenous peptides that signal through Gαq/11-protein coupled receptors (GPCRs) to produce nociceptor sensitization and pain. Both peptides activate phospholipase C to stimulate Ca2+ accumulation, diacylglycerol production, and protein kinase C activation and are rapidly desensitized via a G-protein receptor kinase 2-dependent mechanism. However, ET-1 produces a greater response and longer lasting nocifensive behavior than BK in multiple models, indicating a potentially divergent signaling mechanism in primary afferent sensory neurons. Using cultured sensory neurons, we demonstrate significant differences in both Ca2+ influx and Ca2+ release from intracellular stores following ET-1 and BK treatments. As intracellular store depletion may contribute to the regulation of other signaling cascades downstream of GPCRs, we concentrated our investigation on store-operated Ca2+ channels. Using pharmacological approaches, we identified transient receptor potential canonical channel 3 (TRPC3) as a dominant contributor to Ca2+ influx subsequent to ET-1 treatment. On the other hand, BK treatment stimulated Orai1 activation, with only minor input from TRPC3. Taken together, data presented here suggest that ET-1 signaling targets TRPC3, generating a prolonged Ca2+ signal that perpetuates nocifensive responses. In contrast, Orai1 dominates as the downstream target of BK receptor activation and results in transient intracellular Ca2+ increases and abridged nocifensive responses.

Review on the Current State of Diacylglycerol Production Using Enzymatic Approach

Enzymatic production of diacylglycerol (DAG)-enriched oil has been investigated extensively due to its health benefits with total annual sales of approximately USD 200 million in Japan since its introduction in the late 1990s till 2009. Enzymatic catalysis had been proven to exhibit improved results with respect to yield, purity, reaction time, and stability in comparison with chemical catalysis. The cost of the enzymes, however, is the main hurdle to the widespread use of enzyme for commercial DAG production. This paper attempts to review and summarize various lipase-mediated technological methods for DAG production. Critical aspects such as process considerations on DAG synthesis, mass transfer limitations as well as kinetic mechanism models developed for each enzymatic approach in DAG synthesis are also presented and discussed. In addition, possible reactor configurations were evaluated, if lipase-assisted DAG production is to be technically and economically feasible at an industrial scale.

Differential scanning calorimetry study on caprylins

Monoacylglycerols (MAG) and diacylglycerols (DAG) are nonionic surfactants with several applications in the food, pharmaceutical and other chemical industries. They can be produced by alcoholysis, esterification or partial hydrolysis of triacylglycerols, alternatively using lipase instead of chemical catalysts, generating purer and less degraded lipids. Differential scanning calorimetry (DSC) is a widespread method extensively used for studying the thermal properties of several materials, including the various classes of lipids. In this field, however, the use of DSC has generally been limited to the analysis of the thermal profiles of fats and oils, as well as the characteristics of interesterification reaction products. Therefore, the present study reports the melting and crystallization behavior of chromatographic standards of monocaprylin (MC), dicaprylin (DC) and tricaprylin (TC), as well as binary mixtures of MC and DC in proportions (w/w) of 2:1, 1:1 and 1:2, respectively, aiming to propose the use of DSC as a first step to monitor esterification or hydrolysis reactions for the production of MAG and DAG, as well as the subsequent indispensable purification procedures. It is worth to highlight that little is known about the physical behavior of MAG and DAG of caprylic acid presented herein. The remarkable differences between the behaviors of pure components and binary systems during cooling and heating can be possibly attributed to interactions between molecules by weak chemical bonds such as hydrogen bonds, van der Waals force or hydrophobic interactions, as well as phase transitions or polymorphism. This study indicates that DSC can be a valuable tool to supply important information on catalytic reaction evolution, which should be complemented by other techniques to provide a complete depiction of the reaction under research.

Hydrolysis of soybean oil to produce diacylglycerol by a lipase from Rhizopus oryzae

A lipase from Rhizopus oryzae (rProROL) was produced by recombinant Pichia pastoris in high productivity with a maximum lipase activity of 15,900U/mL, and was employed to hydrolyze soybean oil for the production of diacylglycerol (DAG).Changes in triacylglycerol (TAG) conversion and DAG content were observed under various factors, like pH, water content, temperature and enzyme loading. A polynomial equation (R2=0.9797) between DAG content and TAG conversion was modeled and showed good agreement with the experimental results. The highest content of DAG at 32.09% was observed when TAG conversion was between 68% and 73%. Besides, 13C nuclear magnetic resonance (NMR) was used to identify the 1,2(2,3)-DAG and 1,3-DAG, and during hydrolysis, rProROL was discovered to show 1,3-regioselectivity in the hydrolysis. Moreover, rProROL exhibited high hydrolytic activity and capacity in the accumulation of DAG, indicating that rProROL is a prospective enzyme which could be used in the oils and fats industries.