Interesterification and hydrolysis catalyzed by fatty acid‐modified lipases

Native lipases often exhibit poor interesterification activity. We previously developed a fatty acid modification method to improve the activity of lipases. In this study, we applied this fatty acid modification method to several lipases and evaluated their interesterification and hydrolytic activities. The resulting interesterification activity was strongly dependent on the modifying fatty acid used. Of the saturated fatty acids tested, stearic acid modification substantially improved the interesterification activity of three lipases. Hydrolytic activity was affected slightly by the modifying fatty acid used. Substrate specificity of the modified lipase with triglycerides was also investigated and it was found that fatty acid modification changed the substrate specificity of some lipases.     

Enzymatic enantioselective hydrolysis of acyloxyferrocenophanes using lipases and esterases

Enantioselective hydrolysis of racemic acetate or butyrates of 1-hydroxy [3](1,1′) ferrocenophane and endo-1-hydroxy [4](1,2) ferrocenophane using lipases, pig liver esterase and horse liver esterase resulted in the formation of (R)-alcohols and (S)-esters.     

Triolein-phosphatidylcholine-cholesterol emulsions as substrates for lipoprotein and hepatic lipases

Lipolysis of emulsified glycerol tri[9,10-³H]oleate by lipoprotein lipase purified from bovine milk (E.C. 3.1.1.34) and by hepatic lipase purified from rat liver perfusate was studied as a function of the phosphatidylcholine molecular species and the cholesterol content of the emulsions. Overall, the activities of the two enzymes were similar on a molar basis. Lipoprotein lipase initial lipolysis rates also were comparable for emulsions made with egg phosphatidylcholine or with saturated (dimyristoyl, dipalmitoyl and distearoyl) phosphatidylcholines when cholesterol was low. Increasing the cholesterol content of the emulsion from 2–3 mole percent to 7–14 mole percent reduced triolein lipolysis by lipoprotein lipase in emulsions made with saturated phosphatidylcholines. Rat hepatic lipase was more sensitive to increased cholesterol in emulsions made with saturated phosphatidylcholines than was lipoprotein lipase. The ability to maintain triolein lipolysis during longer incubations differed strikingly among the emulsions and for the two enzymes. Lymph chylomicrons were better substrates for both enzymes than any of the emulsions.     

Surfactant modification of lipases for lipid interesterification and hydrolysis reactions

Commercial grade lipases from Rhizopus japonicus, R. delemar, and Rhizomucor miehei were modified by surfactant (sorbitan monostearate), and their protein recovery and interesterification, and hydrolysis activities were investigated. By repeating the lipase modification processes three times, total protein recoveries of 17–35% could be obtained. The original lipases had no interesterification activities at all; however, all modified lipases in the first process had significant interesterification activities. In the hydrolysis reactions, all modified lipases obtained from the first process showed about three times higher specific activities than the original lipases. The modified lipases obtained from the second and third processes had lower specific interesterification and hydrolysis activities than the lipases from the first one. These results suggest that the surfactant modification process is effective not only for interesterification but also lipase purification.     

Comparative study of commercially available lipases in hydrolysis reaction of phosphatidylcholine

Nineteen 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.     

Enzymatic phosphatidylcholine hydrolysis in organic solvents: An examination of selected commercially available lipases

Eight commercial lipase preparations were examined for the ability to hydrolyze phosphatidylcholine (PC) in hexane solutions. Only the enzymes fromHumicola lanuginosa, Rhizopus delemar andCandida rugosa displayed appreciable activity. Solvent polarity was the largest single factor affecting activity. TheH. lanuginosa sample was most active in polar solvents. TheR. delemar preparation was most active in polar (2-hexamone) and nonpolar (decane) solvents and least active in solvents of intermediate polarity (hexane). The solvent dependence of the activity of theC. rugosa enzyme varied with the ratio of substrate to enzyme. Different degrees of activity were retained by the three enzymes after passive immobilization on Celite, controlled-pore glass, polypropylene and Amberlite XAD-7 resins. No single resin yielded the best retained activity for all three preparations. When examined in 2-octanone, hexane and isooctane, the Celite-immobilizedR. delemar andH. lanuginosa enzymes exhibited highest activity in 2-octanone, while immobilizedC. rugosa was most active in isooctane. The water content at which maximum activity was observed was relatively independent of solvent polarity and the amount of catalyst but was proportional to the amount of PC in the reaction. The retention of activity by immobilizedRhizomucor miehei lipase (Lipozyme) during multiple hydrolytic cycles required a reduction in the water content of the system below that yielding optimal activity in a single cycle.     

Very low density lipoprotein secretion by cultured hepatocytes of rabbits fed purified or autoxidized cholesterol

The main objectives of this study were to compare the effects of dietary commercial cholesterol (containing 5% of oxidized cholesterol derivatives) and purified cholesterol on the secretion rate of very low density lipoprotein apolipoproteins and lipids by cultured rabbit hepatocytes and to verify the hypothesis that products of cholesterol autoxidation stimulate the rapid development of hypercholesterolemia. Rabbits fed dietary (old) commercial cholesterol for six weeks showed a fivefold increase in the serum concentration of cholesterol compared with that in purified cholesterol-fed rabbits. The secretion rates of very low density lipoprotein total protein and very low density lipoprotein [³H]apolipoproteins were similar for the hepatocytes of these two cholesterol-fed groups of animals and were two- and threefold greater, respectively, than for cells from control rabbits. Cholesteryl ester content of the hepatocytes from dietary (old) commercial cholesterol-fed rabbits was dramatically increased in comparison with hepatocytes from control and purified cholesterol-fed rabbits. The elevated intracellular cholesteryl ester content is assumed to account for such an increase of very low density lipoprotein-cholesteryl ester secretion by cells prepared from dietary (old) commercial cholesterol-fed rabbits. These effects appear to be caused by activation of cholesterol esterification by oxidized cholesterol derivatives. The rapid development of hypercholesterolemia induced by dietary (old) commercial cholesterol is associated, at least in part, with the stimulated production of hepatic very low density lipoprotein apolipoproteins and cholesteryl esters.     

Studies on the substrate specificity of purified human milk lipoprotein lipase

The fatty acid specificity of purified human milk lipoprotein lipase was studied using the C₁₈ to C₅₄ (total acyl carbon number) saturated and the C₅₄ mono-, di- and triunsaturated monoacid triacylglycerols. Kinetic determinations indicated that the medium-chain triacylglycerols were better substrates than long- or very short-chain saturated triacylglycerols. The unsaturated triacylglycerols were hydrolyzed at rates comparable to that of tricaprylin with triolein having the highest rate of hydrolysis of the unsaturated species tested. The enzyme attacked the primary ester bond much more readily than the secondary ester bond. The purified human milk lipoprotein lipase showed a preferential stereospecific lipolysis of thesn-1-position of the triacylglycerol molecule.     

Polyunsaturated fatty glyceride syntheses by microbial lipases

The degree of glyceride syntheses by lipase TOYO (Chromobacterium viscosum) and lipase OF (Candida cylindracea) using individual free fatty acids C_18∶1, C_18∶2, C_18∶3, C_18∶4, C_20∶4, C_20∶5 and C_22∶6 were compared. Lipase TOYO incorporated each of the fatty acids into glycerol at levels of greater than 89%. Lipase OF incorporated most of the fatty acids at levels above 70% (docosahexaenoic acid incorporation was 63%). It was concluded that these two lipases are feasible for producing glycerides from unsaturated fatty acids.     

Staphylococcal lipases

A lipase rich fraction was isolated from the cell free supernatant of 24 hr broth culture ofStaphylococcus aureus B-120, grown in trypticase soy broth at 37 C. Lipase from the cell free supernatant was precipitated with equal volumes of absolute ethanol. This fraction was purified further by differential precipitation at pH 8.6 and 4.3. Subsequent purification, using Sephadex G-200 and BioGel 300, yielded a preparation with 350–450-fold increase in specific activity. The purified lipase had an optimum pH of 8.5 at 37 C. The electrophoretic mobility was-7.78×10⁻⁵ cm²/volt/sec. The sedimentation coefficient for the two peaks was 2.85 and 8.5, respectively, and the mol wt was 100,000. The purified lipase hydrolyzed a variety of natural oils and fats. The amount of free fatty acids liberated from hydrogenated soybean oil (iodine value<3) was one-third compared to natural oils and fats. Gas chromatographic analysis of hydrolyzed synthetic triglyceride, with palmitic, stearic, and oleic acids at the rac 1, 2, and 3 positions, respectively, indicated that the enzyme was capable of hydrolyzing the glycerolfatty acid bonds at all three positions. The yield was 40% palmitic, 20% stearic, and 39% oleic acids. Formaldehyde, mercaptoethanol, cysteine, glutathione, and terramycin had inhibitory effects upon lipase activity while hydrogen peroxide, streptomycin, and sodium taurocholate had a stimulatory effect upon the activity.     

Serum lipoprotein distribution,flotation rates and protein analysis

Flotation rates of the major S_f 0–12 lowdensity lipoprotein component in human serum may be calculated from ultracentrifuge data utilizing two computer programs. One program calculates a classical moving boundary uncorrected flotation rate by a best fit straight line for the points (1n x_i, ω²t_i). The other program permits correction for concentration dependence and correction to standard reference conditions. Preliminary application of these methods indicates significantly greater flotation rates in normal human females than in males for the 35–49 year age group. The significance of interrelationships between the serum lipoprotein spectra, the serum lipids and the serum proteins is considered, resulting in the development of a revised method of measuring serum proteins by precision refractometry. The refractometric measurement is corrected in accordance with (any of various) lipid measurements in order to account for the contribution of lipoproteins to the total refractive increment. Such a technique, giving potentially a very accurate protein measurements, has application in screening studies involving abnormalities of both serum lipoprotein and serum protein metabolism.     

The hydrolysis of very low density lipoproteins and chylomicrons of intestinal origin by lipoprotein lipase in ruminants

The hydrolysis by lipoprotein lipase of a very low density lipoprotein/chylomicron fraction, obtained from the intestinal lymph of sheep, has been studied in vitro. Rapid hydrolysis of triacylglycerols, with an accumulation of free fatty acids, was observed. After an initial lag period, phosphatidylcholine also was hydrolyzed. No specificity for particular fatty acids in the triacylglycerols (or phosphatidylcholines) was observed.     

Normalization of high density lipoprotein in fish eye disease plasma by purified normal human lecithin: Cholesterol acyltransferase

Plasma from a patient with fish eye disease has been enriched with autologous high density lipoproteins (HDL) and supplemented with highly purified normal human plasma lecithin:cholesterol acyltransferase (LCAT). Incubation of such plasma at 37 C in vitro resulted in normalization of its low HDL cholesteryl ester percentage, from 23% to 79%, associated with a two-fold increase in both the cholesteryl ester and triglyceride contents of the HDL fraction, as compared to incubation experiments with absent or heat-inactivated purified normal LCAT. The normalization of the HDL cholesteryl ester percentage induced by incubation with purified normal LCAT also was accompanied by an increase in the size of the original fish eye disease HDL particles, which had a mean mass of 115 kd, to HDL particle populations with mean particle masses ranging from 130–220 kd, depending on the concentration of purified LCAT in the incubate. Both HDL cholesterol esterification and particle enlargement were abolished completely by the LCAT inhibitor DTNB and by heat inactivation of the purified normal LCAT. The results give further evidence that fish eye disease is an α-LCAT deficiency.     

Covalent inhibition of digestive lipases by chiral phosphonates

Designing and synthesizing specific inhibitors is of fundamental value for understanding the molecular mechanisms involved in the interfacial adsorption step as well as the catalytic activity of lipases. In this Account, we will review and discuss results obtained mostly at our laboratory concerning the covalent inhibition of human gastric and human pancreatic lipases by chiral phosphonates. Rather than presenting an exhaustive list of compounds tested so far with lipases of animal and microbial origin, we selected recent experimental data illustrating well the specific problems encountered during the covalent inhibition of these digestive lipases.     

Specificity of digestive lipases in hydrolysis of wax esters and triglycerides studied in anchovy and other selected fish

The physiological specificity of fat digestion in several species of marine fish was studied by incubating a variety of synthetic and natural lipid substrates in fish intestinal fluid. Wax ester and triglyceride hydrolyses were studied in vivo and in vitro. In vivo feeding studies showed triglyceride hydrolysis and reesterification in the gut occurred 4 times faster than wax ester metabolism. In vitro comparisons of wax and triglyceride lipolysis always showed triglycerides to be hydrolyzed faster than wax esters; however, wide variation in the ratio occurred among different batches of intestinal juice. Ca. 50% of the 2 monoglycerides formed in the lipolytic sequence were hydrolyzed. Esters of lipase resistent fatty acids (20:4 and 20:5) were cleaved faster than normal fatty acid esters (18:2 and 18:3). Two of the species studied, the northern anchovy, Engraulis mordax and the jack mackerel, Trachurus symmetricus, empty lipase(s) into their gall bladders and produce-phospholipid free bile.     

Highly enantioselective biocatalysts by coating immobilized lipases with polyethyleneimine

Polyethyleneimine (PEI) modification on immobilized lipases greatly enhanced their enantioselectivity in the kinetic resolution of (±)-2-hydroxy-phenylacetic acid methyl ester. The enantiomeric ratio (E) of CNBr-agarose-CRL rose from 8 (without coating) to 20 (ee = 90%) after PEI coating in the hydrolysis at pH 5. In the case of CAL-B, the coating highly improved the enantioselectivity of the immobilized lipase from E = 1.5 (without coating) to E > 100 (ee > 99%). Moreover, this coating strategy improved the stability of the biocatalyst at high temperatures and in the presence of high co-solvent concentrations.     

Biocatalytic synthesis of some chiral pharmaceutical intermediates by lipases

Chiral intermediates were prepared by biocatalytic processes for the chemical synthesis of three pharmaceutical drug candidates. These include (i) the synthesis of [(3R-cis)-3-(acetyloxy)-4-phenyl-2-azetidinone2 for the semi-synthesis of paclitaxel (taxol)5, an anticancer compound; (ii) synthesis of chiral (exo,exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimenthanol monoacetate ester9 for the chemoenzymatic preparation of a thromboxane A₂ antagonist; (iii) the enzymatic synthesis ofS-(−) 3-benzylthio-2-methylpropanoic acid, a key chiral intermediate for the synthesis of antihypertensive drugs captopril10 or zofenopril13.     

Characterisation of acylglycerol synthesis in aqueous foams by lipases

Low water systems have become important for organic synthetic reactions catalysed by enzymes. Esterification reactions could be catalysed by lipase in multiphasic reaction media, such as foams, containing excess water. The esterification of glycerol with oleic acid catalysed by lipase in aqueous foams has been characterised. More than 80% of the oleic acid was converted to glycerides within 10 h. Preliminary characterisation of the reaction with respect to time, pH and temperature indicate that acylglycerol synthesis in foam media as similar to hydrolysis by the same lipase. The observed high K_m of glycerol for the esterification reaction may be due to poor surface active properties of glycerol. The unique advantages of foam as a medium to conduct lipase reactions are discussed. © 1999 Society of Chemical Industry     

Concentration of eicosapentaenoic acid by selective esterification using lipases

The aim of this work was to increase the content of EPA in FFA extracts from a commercial oil (43.1% EPA) and from Phaeodactylum tricornutum oil, a single-cell oil, by selective enzymatic esterification. Initially, the FFA extract was esterified with lauryl alcohol using nine lipases. All the lipases concentrated EPA in the unesterified FFA fraction. The criterion used to choose the best lipase was maximization of the dimensionless effectiveness factor (F_AE). This factor grouped the concentration factor (ratio between the EPA concentrations in the FFA fractions before and after esterification) with EPA recovery in the final FFA fraction. Experiments were carried out to correlate F_AE and the degree of esterification (ED, percentage of initial FA converted to lauryl esters). Lipase AK from Pseudomonas fluorescens was the most effective for concentrating EPA. Studies, of the optimal temperature, substrate molar ratio, solvent/substrate ratio, and treatment intensity (product of the lipase mass and the reaction time) were also carried out using the lipase. The maximum F_AE was obtained when the ED was 60%: EPA concentration was 72%, and recovery was 73%. Finally, this lipase was used to concentrate EPA from a FFA extract from P. tricornutum (23% EPA). The content of EPA in the unesterified FFA fraction increased to 71% at 78% ED (recovery of EPA, 75.5%). Comparison of the results of obtained with the two FFA extracts seemed to indicate that the selectivity of Lipase AK for EPA depended on the content of EPA, with higher contents of EPA in the initial FFA mixture reducing the selectivity for EPA.     

Stereospecificity of different lipases

The stereospecificity of 4 lipases towards enantiomeric alkyldiacylglycerols and alkylmonoacylglycerols was investigated. No stereospecific breakdown of the former substrate was observed for lipases from pancreas,Rhizopus arrhizus, Pseudomonas fluorescens, or bile salt-stimulated lipase from human milk. All lipases degraded 2-oleoyl-3-tetradecyl-sn-glycerol faster than 1-tetradecyl-2-oleoyl-sn-glycerol. Among X-1-acyl-3-alkylglycerol isomers, 1-acyl-3-alkyl-sn-glycerol was preferentially attacked by the 3 first mentioned lipases. Possible mechanisms and metabolic implications for these stereospecificities are discussed. A preliminary account of this work was given in Abstract 457 of the ISF/AOCS World Congress, New York, 1980.