Acidolysis between Triolein and Short-Chain Fatty Acid by Lipase in Organic Solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 °C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.     

Effects of aging on functional properties of caprine milk made into Cheddar- and Colby-like cheeses

The effects of cheese milk obtained at three times during lactation (weeks 4–5, 12–15, and 21–23) and cheese storage (up to 16 or 24 weeks) on meltability, sliceability, and color changes upon heating (232 °C for 5 min, high baking temperature, HT, or 130 °C for 75 min, low baking temperature, LT) of caprine milk cheeses were evaluated. The cheeses were manufactured from milk from Alpine goats and based on the procedures of Cheddar and Colby cheese manufacture. In Cheddar-like cheese, the sliceability (force required to slice sample) was at its highest when the cheese was made with milk from weeks 12–15 into lactation. Color change was variable although it tended to be lowest in cheese made at weeks 4–5 into lactation. In Colby-like cheeses, meltability was at its highest and sliceability was very poor (after 8 weeks of aging) when made with milk obtained later in lactation. Color changes were variable at the two different baking temperatures. As expected during aging, the meltability of the cheeses increased and the force required to slice the cheeses decreased with the significant changes occurring within the first 16 weeks for Cheddar-like and the first 8 weeks for Colby-like cheeses. The color changes upon heating were variable for aged Cheddar-like cheeses and did not change significantly for aged Colby-like cheeses. Color changes were highly correlated with proteolysis occurring during storage. Cheese milk obtained at different times of lactation and aging of the cheese impact the functional properties of caprine milk cheeses and will affect their optimal utilization.     

Biotechnological applications of Candida antarctica lipase A: State-of-the-art

The yeast Candida antarctica produces two different lipases, lipases A and B. While lipase B (CAL-B) is probably the mostly employed hydrolase in the biocatalysis field, the use of the lipase A (CAL-A) has been rather scarce and consequently its tridimensional structure has not been elucidated yet. However, CAL-A is a useful biocatalyst with many different applications that have been described especially in the last few years. Its attractiveness results from its unique features among hydrolases: the high thermostability, allowing operation at T > 90 °C; the ability to accept tertiary and sterically hindered alcohols, which has recently been attributed to the existence of a specific aminoacidic sequence in the active site; the sn-2 recognition in hydrolysis of triglycerides; the selectivity towards trans-fatty acids; the stability in the acidic pH range. Furthermore, it is considered to be an excellent biocatalyst for the asymmetric synthesis of amino acids/amino esters, due to its chemoselectivity towards amine groups. Considering all these aspects, in the present review, the origin, the properties and the applications of the CAL-A are briefly described and discussed, pointing out the unique characteristics of this biocatalyst.     

Long term incubation of cardiac myocytes with oleic acid and very-low density lipoprotein reduces heparin-releasable lipoprotein lipase activity

An exogenous [³H]triolein emulsion was hydrolyzed by intact cardiac myocytes with functional LPL located on the cell surface. This surface-bound LPL could be released into the medium when cardiac myocytes were incubated with heparin. Incubation of cardiac myocytes with VLDL, or the products of TG breakdown, oleic acid or 2-monoolein, did not increase LPL activity in the medium. However, incubation of cardiac myocytes with either VLDL or oleic acid for > 60 min did reduce heparin-releasable LPL activity. In the heart, this inhibitory effect of FFA could regulate the translocation of LPL from its site of synthesis in the cardiac myocyte to its functional site at the capillary endothelium.Abbreviations LPL lipoprotein lipase - TG triacylglycerol - FFA free fatty acids - VLDL very-low density lipoprotein     

SENSORIAL CHARACTERISTICS DURING RIPENING OF THE MURCIA AL VINO GOAT'S MILK CHEESE: THE EFFECT OF THE TYPE OF COAGULANT USED AND THE SIZE OF THE CHEESE

The aim of the present work was to study the sensorial characteristics of Murcia al Vino cheese made with different coagulants (animal rennet and powdered vegetable coagulant obtained from cardoon [Cynara cardunculus]) and of different weights (1 and 3 kg). The cheeses made with rennet showed less odor and taste intensities, a bit clearer color and were harder and grainier, but less creamy than the cheeses made with vegetable coagulant. The big cheeses were characterized by high wine odor, acid odor, wine taste, bitter taste, acid taste, and number, size and distribution of eyes notes. The small cheeses were distinguished by a high overall taste intensity, hardness, overall odor intensity, color, saltiness and graininess. Most of the sensorial characteristics were affected by the ripening time of the cheeses.     

Placental triglyceride accumulation in maternal type 1 diabetes is associated with increased lipase gene expression

Maternal diabetes can cause fetal macrosomia and increased risk of obesity, diabetes, and cardiovascular disease in adulthood of the offspring. Although increased transplacental lipid transport could be involved, the impact of maternal type 1 diabetes on molecular mechanisms for lipid transport in placenta is largely unknown. To examine whether maternal type 1 diabetes affects placental lipid metabolism, we measured lipids and mRNA expression of lipase-encoding genes in placentas from women with type 1 diabetes (n = 27) and a control group (n = 21). The placental triglyceride (TG) concentration and mRNA expression of endothelial lipase (EL) and hormone-sensitive lipase (HSL) were increased in placentas from women with diabetes. The differences were more pronounced in women with diabetes and suboptimal metabolic control than in women with diabetes and good metabolic control. Placental mRNA expression of lipoprotein lipase and lysosomal lipase were similar in women with diabetes and the control group. Immunohistochemistry showed EL protein in syncytiotrophoblasts facing the maternal blood and endothelial cells facing the fetal blood in placentas from both normal women and women with diabetes. These results suggest that maternal type 1 diabetes is associated with TG accumulation and increased EL and HSL gene expression in placenta and that optimal metabolic control reduces these effects.     

Kinetic resolution of amino acid esters catalyzed by lipases

Racemic amino acids were resolved by lipase via hydrolysis of their esters. Lipases (Pseudomonas lipase from Amano PS, Rhizopus lipase from Serva, and porcine pancrease lipase from Sigma) could selectively hydrolyze the L-amino acid esters in aqueous solution with high reactivities and selectivities. The effect of the structural changes in the ester moiety on the stereoselectivity of the lipases was also investigated using D ,L -homophenylalanine as a model. Procedures were developed for the resolution of natural and unnatural amino acids. © 1996 Wiley-Liss, Inc.     

Defined starter system including a bacteriocin producer for the enhancement of cheese flavour

Defined starter systems, consisting of bacteriocin-tolerant Lactococcus lactis subsp. lactis H6 alone or in combination with bacteriocin-sensitive L. lactis subsp. cremoris H1, and low amounts of a bacteriocin-producing culture, were developed and used for the manufacture of semi-hard cheese. Aminopeptidase activity and proteolysis were increased and acidification retarded in cheeses made from milk inoculated with lactococci and the bacteriocin-producing culture with respect to cheeses from milk inoculated with only lactococci. Cheeses made with a defined-strain starter system consisting of L. lactis subsp. lactis H6 and the bacteriocin-producing culture received the highest scores for flavour intensity and quality.     

Evaluation of amino acid-decarboxylative microbiota throughout the ripening of an Italian PDO cheese produced using different manufacturing practices

Aim:  To investigate the presence of biogenic amines (BAs) in Montasio cheese produced by using different cheese manufacturing practices.
Methods and Results:  Three batches of Montasio cheese were made in the following way: batch A using raw milk and natural milk culture, batch B with thermized milk and natural milk culture and batch C with thermized milk and natural milk culture added of a commercial starter culture. During 120 days of ripening analyses were performed for microbial counts and BA content; indeed, the potential to produce BAs was screened in lactic acid bacteria and Enterobacteriaceae isolates. At the end of ripening, the total BA contents of cheeses from batches A, B and C were 166·3, 207·3 and 29·8 mg kg⁻¹, respectively. Amino acid decarboxylase activity was widespread among isolates.
Conclusions:  The BA content of Montasio cheese from the three batches was below the threshold proposed as potentially toxic. The highest BA content was found in cheese produced using thermized milk and natural milk culture; therefore, the thermal treatment of milk was not enough by itself to reduce the counts of decarboxylase-positive bacteria in cheese. The use of selected starters guaranteed a low BA content in Montasio cheese.
Significance and Impact of the Study:  The study of the effects of some technological processes on the incidence of decarboxylative microbiota in 'protected denomination of origin' cheeses could provide useful information on the hygienic risk related to their production.     

Glucose‐dependent Insulin Modulation of Oscillatory Lipolysis in Perifused Rat Adipocytes

Objective: We showed glucose‐dependent lipolytic oscillations in adipocytes that are modulated by free fatty acids (FFAs). We hypothesized that the oscillations are driven by oscillatory glucose metabolism that leads to oscillatory formation of α‐glycerophosphate (α‐GP), oscillatory removal of long‐chain coenzyme A (LC‐CoA) by α‐GP to form triglycerides, and oscillatory relief of LC‐CoA inhibition of triglyceride lipases. This study examined the effect of insulin on this hypothesis. Research Methods and Procedures: Samples were collected every minute from perifused rat adipocytes during the basal state followed by insulin (±glucose) or isoproterenol (±insulin; n = 4 each). Results: Insulin caused a significant increase in glycerol release (18%), with a concomitant significant decrease in FFA release (38%). Without glucose, insulin had no effect on glycerol release while still decreasing FFA release (35%). Insulin (5 μU/mL) attenuated the response of lipolysis to isoproterenol (～3‐fold increase with isoproterenol vs. 2‐fold increase with insulin + isoproterenol). However, 1 mU/mL insulin amplified the lipolytic response (～5‐fold increase in glycerol release with insulin + isoproterenol), with a concomitant increase in FFA reesterification (no increase in FFA release compared with isoproterenol alone). Discussion: We interpret these results to be due to insulin's ability to increase glucose uptake and conversion to α‐GP, thus removing LC‐CoA inhibition of triglyceride lipases. While the physiological importance of lipolytic oscillations remains to be determined, we hypothesize that such an oscillation may play an important role in the delivery of FFAs to the liver, β cells, and other tissues.     

Surfactant-Modified lipase for the catalysis of the interesterification of triglycerides and fatty acids

The lipase-catalyzed intresterification of triglycerides and fatty acids in n-hexane was studied. Initially, lipase Saiken was modified with a surfactant of sorbitan esters so that its dispersibility in hydrophobic organic media was improved. The surfactant-modified lipase formed in the modification process carried out in a buffer solution has 1,3-positional specificity and predominantly catalyzed the interesterification reaction in a microaqueous n-hexane system. The modification technique converted inactive lipases to very active biocatalysts for the interesterification of triglycerides and fatty acids. The pH and the weight ratio of surfactant to enzyme used during the lipase modification process have shown significant effects in determining the recoveries of the protein and enzyme activity from the buffer solution, the protein content of the modified lipase complex after being freeze dried, and the interesterification activity of the complex. The water content in the reaction solution has strongly influenced the enzyme activity as well as the distribution of the products. (c) 1995 John Wiley & Sons, Inc.     

Characterization of 1,3-regiospecific lipases from new Pseudomonas and Bacillus isolates

Lipase producing ability of 120 bacterial isolates was examined qualitatively, resulting in 32 lipase producers, which were further screened for 1,3-regiospecificity. Three Bacillus (GK-8, GK-31 and GK-42) and one Pseudomonas (GK-80) were found to produce 1,3-regiospecific lipases. These lipases were alkaline in nature as they showed pH optima of 9.0 and high stability in the alkaline pH range of 8.0–11.0. The lipases from three Bacillus isolates, viz. GK-8, GK-31 and GK-42 showed temperature optima of 37 °C, whereas the Pseudomonas (GK-80) lipase showed optimum activity at 50 °C. The lipase of GK-8 was highly stable and showed enhanced activity in different organic solvents like petroleum ether (172%), diethyl ether (143%) and acetone (135%).     

Crab digestive lipase acting at high temperature: purification and biochemical characterization

In recent years, recovery and characterization of enzymes from fish and aquatic invertebrates have taken place and this had led to the emergence of some interesting new applications of these enzymes. However, much less is known about lipases from crustaceans. A lipolytic activity was located in the crab digestive glands (hepatopancreas), from which a crab digestive lipase (CDL) was purified. Pure CDL has a molecular mass of 65kDa as determined by SDS/PAGE analysis. Unlike known digestive lipases, CDL displayed its maximal activity on long and short-chain triacylglycerols at a temperature of 60 degrees C. A specific activity of 500U/mg or 130U/mg was obtained with TC(4) or olive oil as substrate, respectively. Only 10% of the maximal activity was detected at 37 degrees C. The enzyme retained 80% of its maximal activity when incubated during 10 min at 60 degrees C, and was completely inactivated at a temperature higher than 65 degrees C. Interestingly, neither colipase, nor bile salts were detected in the crab hepatopancreas. Which suggests that colipase evolved in invertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produce bile salts. No similarity between the 13 N-terminal amino acid residues of CDL was found with those of known other digestive lipases.     

Relationships of Plasma Leptin Levels to Changes in Plasma Free Fatty Acids in Women Who Are Lean and Women Who Are Abdominally Obese

HENNES, MAGDA MI, ARNAVAZ DUA, DIANA L MAAS, GABRIELE E SONNENBERG, GLENN R KRAKOWER, AHMED H KISSEBAH. Relationships of plasma leptin levels to changes in plasma free fatty acids in women who are lean and women who are abdominally obese. Regulation of leptin production by the hormonal and metabolic milieu is poorly understood. Because abdominal obesity is commonly associated with elevated plasma free fatty acid (FFA) flux, we examined the effects of augmenting FFA on plasma leptin levels in women who were lean and of suppressing FFA in women with abdominal obesity. In study 1, nine subjects who were lean, after a 12-hour overnight fast, received either intravenous saline or Intralipid plus heparin to increase the plasma FFA concentration to approximately 1000 μmol/ L. After 3 hours of additional fasting, subjects underwent 3-hour hyperglycemic clamps. In study 2, seven subjects with abdominal obesity were evaluated by a similar protocol, but lipolysis and plasma FFA flux were instead maximally suppressed by acipimox. In the individuals who were lean, leptin levels were unchanged during clamping. Increasing plasma FFA reduced plasma leptin from 7.66 ± 0.66 to 7.05 ±0 0.66 (p=0.03), but 3 hours of hyperglycemia plus hyperinsulinemia had no additional effect on leptin levels (7.15 ± 0.71). Basal leptin levels, 4-fold higher in the subjects with obesity, were reduced from 34.6 ± 2.4 μg/L to 32.3 ± 1.1 μg/L (p=0.004) during the clamp period. When plasma FFA flux was suppressed, however, plasma leptin levels after clamped hyperglycemia/hyperinsulinemia were increased to 38.9 ± 1.2 μg/L (p=0.014 vs. time 0 and 0.001 vs. saline protocol). Changes in leptin concentrations are not correlated with changes in FFA. These results suggest that plasma FFA concentration does not regulate plasma leptin levels in basal, extended fasting, or hyperglycemic/hyperinsulinemic states.     

Lipase-catalysed synthesis of glucose fatty acid esters in tert-butanol

Synthesis of 6-O-acylate--d-glycopyranose from underivatised substrates in anhydrous tert-butanol was achieved using immobilised lipases from Candida antarctica and Mucor miehei. Except for acetic acid, the initial reaction rates with the C. antarctica lipase were independent of acyl donor chain lengths and in a range of 3.9±0.4 mol glucose converted min–1 g enzyme preparation. The catalytic activity of the M. miehei lipase increased with increasing acyl donor chain length with a maximum for stearic acid of 0.45 mol min–1 g. Using maltose as substrate, the catalytic activity decreased by a factor of 48 and 20 with the lipase from C. antarctica and M. miehei, respectively, while with maltotriose no reaction was observed.     

Lipase-Catalyzed Regioselective Acylation and Deacylation of Glucose Derivatives

In the development of an efficient synthesis of 1-O-decanoyl-2,3,4,6-tetra-O-acetyl-β-D-glucose (β-2) several lipase-based approaches have been explored. Among five immobilized Upases tested, the lipase from Candida antarctica proved particularly efficient for catalyzing selective hydrolysis in the 1-position of 1,2,3,4,6-penta-O-acetyl-β-D-glucose (β-1). Using triethylamine as catalyst, the hydrolysis product 2,3,4,6-tetra-O-acetyl-D-glucose (3) can be esterified with decanoyl chloride to form β-2 selectively, thereby providing an efficient chemo-enzymatic synthesis starting from readily available raw materials. Attempts to produce β-2 from β-1 by lipase-catalyzed interesterification or to esterify 3 with decanoic acid using a lipase as catalyst were unsuccessful. The latter finding was explained by the hemiacetal OH group of glucose being unable to act as nucleophile in the lysis of the lipase acyl-enzyme intermediate. Furthermore, β-2 was found to bee a too bulky substrate to fit into the active site of any of the lipases tested.     

Effect of water activity and immobilization on fatty acid selectivity for esterification reactions mediated by lipases

The effect of water activity (a(w)) and immobilization on fatty acid (FA) selectivity of Burkholderia (formerly Pseudomonas) cepacia, Rhizomucor miehei, Candida antarctica (type B), and Candida rugosa lipases in esterification reactions was determined. Studies were based on measuring ester formation in multicompetitive reaction mixtures containing either the homologous series of even carbon number n-chain saturated FA (C4-C18) or a series of n-chain (un)saturated FA (C18:X, where X = 0-3 double bonds) as cosubstrates with 1,3-propanediol in ter-butyl methyl ether at a(w) of 0.19, 0.69, and 0.90. Activity and FA selectively patterns were similar for free and Celite-adsorbed lipases in response to changes in a(w'), although specific effects were observed for selectivity of B. cepacia and C. rugosa lipases toward C16 and C4/C6 FA, respectively. Also, selectivity toward unsaturated C18:X FA as a group was modulated by changes in a(w) for three of the four lipase studied. Resin-fixed lipases from R. miehei and C. antarctica exhibited profound differences in activity and FA selectively in response to changes in a(w'), relative to free and Celite-bound forms. These findings suggest that FA selectivity for lipid modification is influenced by a(w) and immobilization, but that each lipase has a characteristic response to these factors in a manner that cannot be predicted.     

Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7

Extracellular thermostable lipase produced by the thermophilic Bacillus stearothermophilus MC 7 was purified to 19.25-fold with 10.2% recovery. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to be 62 500 Da. The purified enzyme expressed maximum activity at 75–80 °C and its half life was 30 min at 70 °C. The K_m and V_max were calculated to be, respectively, 0.33 mM and 188 μM min⁻¹ mg⁻¹ with p-nitrophenyl palmitate (pNPP) as a substrate. Enzyme activity was inhibited by divalent ions of heavy metals, thiol and serine inhibitors, whereas calcium ion stimulated its activity. The most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose. The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well.     

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples.