Nonspecific Lipase Research Articles

Effect of enzymatic transesterification on the melting points of palm stearin‐sunflower oil mixtures

AbstractTransesterification with lipases may be used to convert mixtures of fats to plastic fats, making them more suitable for use in edible products. In our study, 1,3‐specific (Aspergillus niger, Mucor javanicus, Rhizomucor miehei, Rhizopus javanicus, and Rhizopus niveus) and nonspecific (Pseudomonas sp. and Candida rugosa) lipases were used to transesterify mixtures of palm stearin and sunflower oil (PS‐SO) at a 40:60 ratio in a solvent‐free medium. The transesterified mixtures of PS‐SO were analyzed for their percentage free fatty acids (FFA), degree and rate of transesterification, solid fat content, slip melting point (SMP), and melting characteristics by differential scanning calorimetry. Results indicated that Pseudomonas sp. lipase produced the highest degree (77.3%) and rate (50.0 h−1) of transesterification, followed by R. miehei lipase at 32.7% and 27.1 h−1, respectively. The highest percentage FFA liberated was also in the reaction mixtures catalyzed by Pseudomonas sp. (2.5%) lipase and R. miehei (2.4%). Pseudomonas‐catalyzed mixtures produced the biggest drop in SMP (13.5°C) and showed complete melting at below body temperature. All results indicated conversion of the PS‐SO mixtures to a more fluid product. The findings also suggest that the specificity of lipases may not play a significant role in lowering the melting point of the PS‐SO mixtures.

Enzymatic interesterification of triolein and tristearin: Chemical structure and differential scanning calorimetric analysis of the products

AbstractThe structural composition and thermal properties of the products of enzymatic interesterification of triolein and tristearin were investigated. The biocatalyst for the reaction was an immobilized Candida antarctica lipase, SP435. Enzyme load of 10% (w/w reactants) produced 72% of desired total products. Oleoyl‐distearoyl triglycerides (SSO, OSS) had higher melting points than dioleoyl‐stearoyl triglycerides (OOS, SOO) because the sample contained larger amounts of stearic acid than oleic acid residues. SOS and OSO were hardly produced (0.2 to 1.2%), which indicates that SP435 acted as a nonspecific lipase when catalyzing the interesterification of triolein and tristearin. The maximal yield of OSS and SSO (46.9%) was achieved with a 1.2 mole ratio of triolein to tristearin. As the proportion of tristearin was increased, the production of SOO and OOS decreased, the melting profile of the interesterified triglycerides shifted toward higher melting forms, and the solid fat content increased, indicating formation of hard fats.

Structured lipids: Lipase‐catalyzed interesterification of tricaproin and trilinolein

AbstractStructured lipids were synthesized by interesterification of trilinolein and tricaproin with sn‐1,3‐specific (IM 60) and nonspecific (SP 435) lipases. The interesterification reaction was performed by incubating a 1:2 mole ratio of trilinolein and tricaproin in 3 mL hexane at 45°C for the IM 60 lipase from Rhizomucor miehei, and at 55°C for the SP 435 lipase from Candida antarctica. Reaction products were analyzed by reverse‐phase high‐performance liquid chromatography with an evaporative light‐scattering detector. The fatty acids at the sn‐2 position were identified after pancreatic lipase hydrolysis and analysis with a gas chromatograph. IM 60 lipase produced 53,5 mol% dicaproyllinolein (total carbon number = C33) and 22.2% monocaproyldilinolein (C45). SP 435 lipase produced 41% C33 and 18% C45. When caproic acid was used in place of tricaproin as the acyl donor, the IM 60 lipase produced 62.9% C33. The effects of variation in mole ratio, temperature, added water, solvent polarity, and time course on the interesterification reaction were also investigated. In the absence of organic solvent, IM 60 lipase produced 52.3% C33.

Calcium Modulation of Toxicities Due to Cisplatin

Cisplatin (CDDP) is a potent anti-neoplastic agent with associated toxicities, especially gastrointestinal and nephrotoxicity that are its dose-limiting factors in clinical oncology. In an attempt to elucidate its mechanism(s) of action, liver and kidney tissues from normal and CDDP treated (1.8 mg/kg) dogs were evaluated for changes in various dehydrogenases [MDH, SDH, β-HBDH, IDH and G-6-PDH] and nonspecific lipase enzymes. CDDP treatment induced an inhibition of all the enzymes studied except G-6-PDH and nonspecific lipases, where there was a significant increase. Supplemental pretreatments with calcium 2.50 mg (150,000 USP units) ergocalciferol plus 1000 mg of elemental calcium as Tums 500 (EffeCal; calcium carbonate)/day seemed to retain enzyme levels close to normal with no apparent toxic side effects observed after CDDP. Calcium supplements post-CDDP treatment did not have any protective effect.

Establishment and characterization of murine bone marrow stromal macrophage line

The authors established a murine bone marrow stromal cell line QXMSC1 by discarding the suspensible hematopoietic cells and passaging the adherent stromal cells many times in long-term bone marrow culture. QXMSC1 cells have been passaged 65 times for 15 months and immortalized. The mean number of QXMSC1 chromosomes is 66 ± 4. The appearance of the cells is elliptical. There are many pseudopods and mononuclei under the optical microscope. Many lisosomes and phagosomes in cytoplasm exist under the transmission electron microscope. There are no desmosome junction between the cells, no lipid drops, no intermediate filament. Vimentin is positive and keratin is negative by stain of immunocytochemistry. Nonspecific lipase staining is positive. These results indicate that QXMSC1 cells are murine bone marrow macrophages.

Role of calcium in modulation of toxicities due to cisplatin and its analogs

Cisplatin, carboplatin and paclitaxel (taxol) are potent antineoplastic agents with associated toxicities, especially gastrointestinal and nephrotoxicity that are their dose-limiting factors in clinical oncology. In an attempt to elucidate their mechanism(s) of toxicity, liver and kidney tissues from normal and drug treated rats and dogs were evaluated for changes in various dehydrogenase and non-specific lipase enzymes. Histochemically, cisplatin treatment induced an inhibition of all the enzymes studied except glucose-6-phosphate dehydrogenase and non-specific lipases, where there was a significant increase. Supplemental treatments with calcium [1 ml of 1.3% CaCl2/day in rats and 2.50 mg (150000 USP units) ergocalciferol plus 1000 mg of elemental calcium as TUMS 500 (EffeCal)/day in dogs] seem to protect against severe gastrointestinal toxicity and nephrotoxicity.

Spectrophotometric Determination of the Positional Specificity of Nonspecific and 1,3-Specific Lipases

Using commercially available thiosubstrates, such as 2,3-dimercapto-1-propanol tributyrate, the regiospecificities of 1,3-specific and nonspecific lipases was confirmed. The spectrophotometric test is a simple, rapid, and convenient alternative method to those previously reported for the characterization of the positional specificities of new lipases.

Comparative study of commercially available lipases in hydrolysis reaction of phosphatidylcholine

AbstractNineteen commercial lipase preparations were compared with respect to their ability to hydrolyze phosphatidylcholine (PC) in a reverse micellar system. In terms of the source organism, lipases from Mucor or Rhizopus species showed comparatively high reactivity, while those from Aspergillus and Humicola sp. exhibited little PC hydrolysis activity. Almost no hydrolysis reaction was observed by positionally nonspecific lipases derived from Candida sp. The results suggest that particular attention should be paid to the source organism in selecting lipases for use in the enzymatic conversion of phospholipids.

Production of two types of non-specific lipases by Geotrichum sp. FO 274A:A fish oil-assimilating strain.

Geotrichum sp. FO 274A was isolated as a microorganism capable of assimilating sardine oil as the sole carbon source. Three FO 274A lipases were purified by ion exchange and hydrophobic interaction chromatographies and termed Lipases A, B and C. Lipases A and C hydrolyzed most of the ester bond of the fatty acids contained in sardine oil. Lipase B preferentially hydrolyzed the ester bond of the fatty acids of C 16 or C 18 having the cis double bond at the 9-position. The hydrolysis rate for sardine oil under indicated conditions increased when combinations of the purified lipases were used instead of each purified lipase. A combination of Lipases A and C showed the highest hydrolysis rate for sardine oil. The lipases from Geotrichum sp. FO 274A showed the highest hydrolysis rate for sardine oil compared with the lipases similarly isolated from other Geotrichum sp.

Low‐calorie triglyceride synthesis by lipase‐catalyzed esterification of monoglycerides

AbstractMonoglycerides of erucic acid (C22:1, Δ13), prepared by conventional methods, were reacted with caprylic acid (octanoic acid, C8.0) by using lipases as catalysts with the intention of synthesizing a triglyceride that contains two molecules of caprylic acid and one molecule of erucic acid (caprucin). The reaction was carried out by mixing lipase powder, a small quantity of water, and the reactants in a temperature‐controlled stirred batch reactor. Organic solvents or emulsifying agents were not required. When the nonspecific lipase fromPseudomonas cepacia was used, a yield of approximately 37% caprucin was obtained, together with a complex mixture of di‐ and triglycerides that resulted from the random transesterification of the erucic acid. The fatty acid‐specific lipase fromGeotrichum candidum promoted minimal transesterification of erucic acid and resulted in a yield of 75% caprucin and approximately 10% interesterification products. Lipase fromCandida rugosa exhibited a similar, although less pronounced, specificity to that fromG. candidum and promoted more transesterification of erucic acid. Optimum conditions forG. candidum lipase were at 50°C and an initial water content of 5.5%. After the reaction, erucic acid was converted to behenic acid by hydrogenation, thereby converting caprucin into caprenin, a commercially available low‐calorie triglyceride.

Carica papaya latex lipase:sn‐3 stereoselectivity or short‐chain selectivity? Model chiral triglycerides are removing the ambiguity

AbstractShort‐chain fatty acids are usually located at positionsn‐3 in natural triglycerides, particulary in dairy fats. As a result, it is extremely difficult to differentiate betweensn‐3 stereospecificity and short‐chain typoselectivity in many lipases and acyltransferases that perform in this way. This ambiguity can be removed through successive use of a chiral triglyceride with a short fatty acid in positionsn‐1 and of its racemic in controlled hydrolysis reactions. After checking that the proposed method effectively confirmed the type of activity of control biocatalysts (Candida cylindracea nonspecific lipase andMucor miehei 1–3 regiospecific lipase), we confirmed thatCarica papaya latex has a strictsn‐3 stereospecificity.

Lymphatic fatty acids from rats fed human milk and formula containing coconut oil

Human milk and infant formula containing coconut/soy oil were infused into the duodenum of rats to determine the incorporation of capric, lauric, myristic and palmitic acids into lymphatic triacylglycerol (TAG). The proportion of capric and lauric acids in the lymphatic TAG reflected the fatty acid composition of the diet. Based on positional analysis, it appears that more than 50% of the capric and lauric acids could have been absorbed from the intestine as sn-2 monoacylglycerols. In the rats fed human milk, 50% of palmitic acid in lymphatic TAG was in the sn-2 position. Because of the nonrandom distribution of palmitic acid in the lymphatic TAG, the nonspecific lipase in human milk, i.e., bile salt-stimulated lipase, did not appear to be a factor in milk lipid digestion.

Signal transduction for taurocholic acid in the olfactory system of Atlantic salmon.

Conjugated bile acids such as taurocholic acid (TChA) are potent olfactory stimuli for Atlantic salmon (Salmo salar). A plasma membrane rich fraction was derived from salmon olfactory rosettes and used to investigate TChA signal transduction and receptor binding. In the presence of GTP gamma S, TChA caused dose-dependent stimulation of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown, half maximal at less than 10(-7) M TChA. Stimulation of PIP2 breakdown by TChA required GTP gamma S, was blocked by GDP beta S, and was mimicked by A1F4-, consistent with a G protein requirement. A1F4- and Ca2+ stimulated breakdown of PIP2, but not phosphatidylcholine, arguing against a non-specific lipase activation. Stimulation of PIP2 breakdown by TChA was maximal at low Ca2+ concentration, < or = 10 nM. Conventional binding analysis with 3H-TChA was inconclusive due to a high degree of non-specific binding and to lack of tissue specificity expected for an olfactory receptor. Analysis of odorant amino acid binding indicated possible interaction of TChA with a putative acidic amino acid receptor but no interaction of TChA with a putative neutral amino acid receptor. We conclude that olfactory discrimination between amino acids and bile acids occurs in part at the receptor level while both classes of odors appear to use the same signal transduction mechanism, G protein mediated activation of phosphoinositide specific phospholipase C (PLC).

Enzymatic pretreatment of deodorizer distillate for concentration of sterols and tocopherols

Separation of sterols and tocopherols from fatty acids in deodorizer distillate was facilitated through lipase‐catalyzed modification of fatty acids in canola, mixed and soya deodorizer distillates. The fatty acid esterification with methanol catalyzed by SP‐382 (an immobilized nonspecific lipase) proceeded rapidly, with conversion of fatty acid to methyl ester in 5 h being 96.5, 83.5 and 89.4%, respectively. A model mixture of pure oleic acid and dl‐α‐tocopherol was used to study any potential side reactions that may lower the tocopherol content during the esterification reaction. Under the conditions employed, the loss of tocopherol was less than 5%. Simple vacuum distillation (1–2 mm Hg) was employed to remove the volatile fraction (methyl esters of fatty acids, some fatty acids and other volatiles) of the esterified deodorizer distillate, leaving behind sterols, sterol esters and tocopherols. Sterols and tocopherols were almost completely retained in the residue fraction with recoveries in the range of 95%. Overall recoveries of sterols and tocopherols after esterification and distillation were over 90% for all the deodorizer distillate samples.

Use of specific polyclonal antibodies to detect heterogeneous lipases from Geotrichum candidum

Geotrichum candidum CMICC 335426 was previously shown to produce two lipases termed lipase A and lipase B, lipase B being highly specific for hydrolysis of esters of cis- Δ9 fatty acids. We now describe the isolation of polyclonal antibodies specific for lipase A and lipase B. These antibodies were used in Western blotting techniques to detect the appearance of the lipases during the course of the fermentation of G. candidum CMICC 335426. Lipases A and B were found to be produced simultaneously in the extracellular medium at the start of the growth phase. The two lipases were always present at similar levels in the medium. The specific antibodies were then used to detect the presence of A- and B-like lipases in crude lipase samples from other strains of G. candidum. The lipases were found at different levels in all these samples, and the specificities of the crude lipases varied significantly from one strain to another. Differences in specificity could therefore be explained by different levels of specific (B-type) and non-specific (A-type) lipases in the medium. This was verified by purifying A- and B-type lipases from the G. candidum strain ATCC 34614.

Enzymatic methylation of canola oil deodorizer distillate

Methylation of canola oil deodorizer distillate catalyzed by a nonspecific lipase was investigated. The conversion of fatty acids to methyl esters has been optimized by using a statistical design. Up to 96.5% conversion of fatty acids to their methyl esters has been achieved without the aid of vacuum or any water‐removing agent. The effects of temperature, ratio of the reactants (methanol: fatty acids in the deodorizer distillate) and enzyme concentration on the equilibrium conversion were studied. The temperature and ratio of the reactants showed a significant effect on the conversion of fatty acids to methyl esters and they exhibited a strong interactive effect. Enzyme concentration in the range of 2.7% to 4.3% did not show a significant effect on the equilibrium conversion of fatty acids. Greater than 95% conversion of fatty acids to methyl esters was achieved at temperatures around 50°C and at a ratio of the reactants between 1.8 and 2.0. The inhibitory effect of hydrophilic methanol on the enzyme activity was largely reduced by working at the lower temperature range (around 50°C).

Effect of lipase specificity on triglyceride synthesis

Enzymatic triglyceride synthesis from free fatty acid and glycerol is shown to be catalysed by both 1,3-specific lipase and non-specific lipase. This paper elucidates the mechanism of the reaction, showing that, with a 1,3-specific lipase, the 1,3-diolein enzymatically formed has to isomerise spontaneously to 1,2-diolein in presence of oleic acid, before being transformed to triolein by the enzyme.

Effect of randomization on oxidative stability of vegetable oils at two different temperatures

Five vegetable oils were each randomized by chemical means (with the use of a sodium potassium alloy) and by enzymatic means (using a nonspecific lipase). The success of the randomization procedure was confirmed via positional analysis. The oxidative stabilities of the native and chemically randomized oils were determined at storage temperatures of 28°C and 55°C using absorbance at 234 nm as indicative of conjugated diene content. No difference between curves occurred in oils stored at 55°C, however, at the lower temperature all chemically randomized oils had significantly steeper slopes (P&lt;0.05), suggesting a lower stability. When both enzymatically and chemically randomized oils were compared to native oils at 28°C, no significant difference occurred between slopes of native and enzymatically randomized oils, however, the end content of conjugated dienes was significantly higher for chemically randomized canola, corn and soybean oils (P&lt;0.05). No difference was seen between the slopes of the three different oils from either linseed or sunflower. Since both of these oils exhibited higher oxidation rates, it is possible that observation of differences between the stability of native and chemically randomized oild is dependent upon the rate of the reaction.

Interesterification and synthesis by [formula omitted] lipase in microemulsions

Unusual reactions of interesterification and synthesis catalyzed by Candida cylindracea lipase have been tested in reverse microemulsions. The microemulsions used are made of fatty acids or triglycerides, the enzyme dissolved in a very low water quantity, Brij 35 used as surfactant and an alcoholic cosurfactant. In such a system, fats and alcohols are both the substrates of the enzyme and the microemulsion components. Incidentally, non specific Candida cylindracea lipase does not catalyze interesterification of short chain triglycerides, revealing a specificity for the chain length. Interesterification reactions tested in the presence of a given water quantity but with varying water activities show that it is the water activity and not the water quantity which is a fundamental parameter of the system. The effect of the surfactant (Brij 35) on the interesterification reaction is studied. Heptyl-oleate synthesis catalyzed by non-specific lipase is obtained in microemulsions at a 98 % yield. Synthesis of glycerol esters is also tested in monophasic medium and mono and diglycerides are obtained.

Discovery of a cutinase-producing Pseudomonas sp. cohabiting with an apparently nitrogen-fixing Corynebacterium sp. in the phyllosphere

A phyllospheric bacterial culture, previously reported to partially replace nitrogen fertilizer (B. R. Patti and A. K. Chandra, Plant Soil 61:419-427, 1981) was found to contain a fluorescent pseudomonas which was identified as Pseudomonas putida and a Corynebacterium sp. The P. putida isolate was found to produce an extracellular cutinase when grown in a medium containing cutin, the polyester structural component of plant cuticle. The Corynebacterium sp. grew on nitrogen-free medium but could not produce cutinase under any induction conditions tested, whereas P. putida could not grow on nitrogen-free medium. When cocultured with the nitrogen-fixing Corynebacterium sp., the P. putida isolate grew in a nitrogen-free medium, suggesting that the former provided fixed N2 for the latter. These results suggest that the two species coexist on the plant surface, with one providing carbon and the other providing reduced nitrogen for their growth. The presence of cutin in the medium induced cutinase production by P. putida. However, unlike the previously studied fungal systems, cutin hydrolysate did not induce cutinase. Thin-layer chromatographic analysis of the products released from labeled apple fruit cutin showed that the extracellular enzyme released all classes of cutin monomers. This enzyme also catalyzed hydrolysis of the model ester substrates, p-nitrophenyl esters of fatty acids, and optimal conditions were determined for a spectrophotometric assay with p-nitrophenyl butyrate as the substrate. It did not hydrolyze triacyl glycerols, indicating that the cutinase activity was not due to a nonspecific lipase. It showed a broad pH optimum between 8.0 and 10.5 with 3H-labeled apple cutin as the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)