Synthesis of medium-chain glycerides by lipase in organic solvent

Using commercial lipases from various microbial origins, medium-chain glycerides, such as mono-, di-, and tricaprin, were synthesized in isooctane from glycerol and capric acid. The enzyme reaction was performed with 0.35 M capric acid, 0.025 M glycerol, and 0.46 g silica gel to remove water in 5 mL of isooctane with 30 mg lyophilized lipase. Of the 21 kinds of lipases, 11 showed good synthetic activities. Lipases fromPseudomonas aeruginosa (Lipase PS),Rhizomucor miehei lipase andChromobacterium viscosum lipase (Lipase CV) showed high activities for the production of tricaprin, while lipase OF-360 (fromCandida rugosa) and lipase D (Rhizopus delemar) were good for dicaprin production. Lipases CC and MY fromC. rugosa (C. cylindracea) and lipase D (Rhizopus delemar) were good for dicaprin production. Lipases CC and MY fromC. rugosa (C. cylindracea) also showed high activities for dicaprin and tricaprin. Some lipases, especially lipase PS, had high thermal stability over 60°C. The optimal lyophilization pH to dehydrate the lipase coincides with the optimal buffer solution pH for hydrolysis.     

固定化假丝酵母脂肪酶催化合成辛酸甘油酯

以自制固定化假丝酵母脂肪酶作为催化剂,研究了无溶剂体系中辛酸和甘油直接酯化合成辛酸甘油酯的反应条件。考察了酶的种类、底物的物质的量之比、温度、酶量、甘油的初始含水量和反应时间等因素对辛酸转化率和产物组成的影响。结果证明,以纺织物作为载体制备的固定化假丝酵母脂肪酶适宜催化辛酸甘油酯的合成。最优反应条件为：辛酸与甘油的物质的量之比为2∶1,固定化假丝酵母脂肪酶加量为0.5g/0.69g甘油,温度为40 ℃,振荡培养箱转速为190 r/min。最优反应条件下辛酸转化率可以达到94%以上,经过简单处理的固定化酶可以重复使用4批。     

Production of medium-chain glycerides by immobilized lipase in a solvent-free system

Enzymatic synthesis of medium- chain glycerides (MCGs) was studied by using capric acid (decanoic acid) and glycerol as substrates for immobilized lipase (Lipozyme^TM) without any solvents or surfactants. Quantitative analysis of the reaction mixture was conducted by using highperformance liquid chromatography (HPLC), which enabled the exact tracing of the capric glyceride synthesis. Oleic acid was also used for comparison. The esterification activity of Lipozyme was determined at 40°C in an open batch reactor; the activities were 400 and 200 units/g for the capric glyceride and oleic glyceride synthesis, respectively. Maximum initial reaction rate was obtained at 50°C for capric and 60°C for oleic glyceride synthesis. The time course of the capric glyceride synthesis was compared in terms of different molar ratios, from which we infer that this enzyme is 1,3- specific, but not absolute, in this esterification reaction. The final conversion was greatly influenced by the methods used to remove water, among which the cold trap method resulted in a noticeable improvement. *To whom correspondence should be addressed at Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology, P.O. Box 150, Chongyang, Seoul 130-650, Korea.     

Continuous synthesis of glycerides by lipase in a microporous membrane bioreactor

A membrane bioreactor was developed for continuous synthesis of glycerides by lipase to overcome the drawbacks associated with the usual operation in an emulsion system. One unit (total area: 726 cm²) of flat, plate-type dialyzer was used as the membrane bioreactor at 40 C. The glycerol solution, containing bacterial lipase and water, was supplied continuously to 1 side of a sheet of microporous polypropylene membrane (strongly hydrophobic) and the effluent was recycled, while undiluted liquid fatty acid (oleic or linoleic) was fed continuously to the opposite side of the membrane and came in contact with a glycerol-water-lipase solution to cause the reaction. The product, glycerides, was obtained at the outlet, in a pure state, with no other phase. Highest conversion (ca. 90%) was obtained when the water content of the glycerol solution was 3–4%. As the accumulation of water produced by the reaction lowered the conversion, molecular sieves in a column that the glycerol solution passed through were used for optimal water content. The reaction could be continued at least for 1 month, yielding a conversion above 70% when 1% CaCl₂ was added in the glycerol solution. The main component of glycerides formed was almost equimolar amounts of mono-and diglycerides.     

Structure of unsaturated glycerides. Analysis by countercurrent distribution and lipase hydrolysis

The structure of highly unsaturated triglycerides isolated from soybean, linseed and safflower oils by countercurrent distribution has been determined by the pancreatic lipase hydrolysis technique. Compositions of positionally distinguishable glyceride isomers for the 5-double bond fraction obtained from safflower oil, the 7-double bond fraction from soybean oil and the 8-double bond fraction from linseed oil agreed with the theoretical values calculated according to the VanderWal-Coleman theory of 1,3-random-2-random distribution. The fatty acid analyses for individual isomers of the unsaturated glycerides demonstrated that positional isomers of glycerides may be present in nonrandom amounts even though the compositionally distinguishable glycerides approximate a random pattern. Presented at the AOCS Meeting in Houston, April 1965. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Synthesis of medium-chain glycerides using lipase from Candida rugosa

Enzymatic synthesis of medium-chain glycerides (MCG) from capric acid and glycerol was studied using lipase from Candida rugosa. The effects of various reaction parameters such as time, molar ratio of substrates (mmol capric acid/mmol glycerol), amount of lipase, type of organic solvents, and initial water activity (a _w ) were studied. The best conditions tested for MCG synthesis at 37°C were, respectively, time, 24 h; molar ratio of substrates, 2.5; and amount of lipase, 100.0 mg. The use of organic solvents greatly influenced the activity of lipase in the synthesis of MCG. Generally, activity of lipase was high in nonpolar solvents with log P values from 3.50 to 4.50, where P is the partition coefficient between water and 1-octanol. The enzymatic synthesis of MCG was preferably carried out at an initial a _w of 0.328, which resulted in maximal yield. Analysis of the products of reaction using gas chromatography showed that lipase from Candida rugosa seemed to produce more dicaprin and tricaprin than monocaprin.     

Lipolysis of trivernolin by pancreatic lipase

Vernolic acid (cis-12,13-epoxy-cis-9-oetadece-noic acid) occurs as the triglyceride in the seed ofVernonia anthelmintica. Incubation of the seed produces a 1,3-divernolin. To determine whether the structure of trivernolin is responsible for the apparent secondary ester position specificity of the natural enzyme, trivernolin and tri-olein, were incubated with pancreatic lipase and the free fatty acids and monoglycerides were determined after 5 and 15 min digestion periods. The preponderance of 2-monoglyceride produced by the action of pancreatic lipase was interpreted to indicate that the structure of trivernolin was not solely responsible for the secondary position specificity of theV. anthelmintica lipase toward trivernolin.     

Immobilization of porcine pancreatic lipase on celite for application in the synthesis of butyl butyrate in a nonaqueous system

For immobilization of lipase, the use of a porous support material is recommended so that suitable amounts of lipase can be spread on a surface area without conformational changes. In this work, porcine pancreatic lipase was deposited on Celite, either by direct binding from aqueous solution or by deposition from aqueous solution by the addition of organic solvent. The influence of the immobilization procedure on the activities of the derivatives has been studied regarding their ability to synthesize butyl butyrate. The reaction rates were compared with the rate of esterification with free lipase. Better properties were displayed when the immobilized lipase form was prepared in an apolar solvent such as hexane. Under suitable reaction conditions, esterification yields as high as 90% were attained. Batch operational stability tests indicated that no enzyme deactivation occurs after 15 consecutive batches of 24 h each.     

Hydrolysis of synthetic triacylglycerols by pancreatic and lipoprotein lipase

The stereochemical course of the hydrolysis of synthetic sn-glycerol-1-palmitate-2-oleate-3-linoleate, sn-glycerol-1,2-dipalmitate-3-oleate and their antipodes by pancreatic and milk lipoprotein lipase was investigated by thin layer and gas liquid chromatographies of the diacylglycerol intermediates. The enzymic hydrolyses were made with bile salts or lysolecithin in a 1∶1 molar ratio to the substrate as emulsifiers and were limited to short time intervals which minimized isomerization and the reversal of lipolysis. In all instances, the products of hydrolysis by lipoprotein lipase contained a marked preponderance of the 2,3-diacylglycerols, while the composition of the diacylglycerol intermediates in the products of pancreatic lipase varied with the nature of the fatty acid in the 1 and 3 positions of the triacylglycerol molecule. Pancreatic lipase, but not lipoprotein lipase, gave a preferential release of unsaturated fatty acids. The above results are similar to those obtained with radioactive trioleoylglycerol and conventional stereospecific analyses and suggest that lipoprotein lipase may favor attack on the sn-1 position. It is hypothesized that the small amounts of the 1,2-diacylglycerols present may have arisen from a reversal of lipolysis also catalyzed by this enzyme. Presented in part at the AOCS Fall Meeting, Ottawa, September 1972.     

A colorimetric assay of pancreatic lipase: Rapid detection of lipase and colipase separated by gel filtration

A rapid assay for pancreatic lipase (E.C., glycerol-ester hydrolase 3.1.1.3) is described. The assay is based on the color change of a pH indicator as butyric acid is released from the substrate tributyrin. A mixture made with tributyrin and the water soluble components of the assay is ideally suited for use as a rapid test as, for example, when assaying chromatography fractions. Quantitative data can be obtained by measuring the disappearance of absorbance at 557 nm versus a blank reaction. The assay has been used in the rapid preparation of colipase-free lipase and colipase.     

Preparation of glycerides by controlled esterification

A simple procedure for esterifying glycerides without interesterification occurring would be highly useful for preparing on a large scale a number of tailor-made fats, including cocoa butter-like fats. Such esterifications were carried out by employingp-toluenesulfonic acid as catalyst and continuously removing the water of esterification by azeotropic distillation with aliphatic hydrocarbons or by stripping with vaporized hydrocarbons. Even thoughp-toluenesulfonic acid rapidly disproportionated 1-monostearin, even at 120C, apparently esterification was faster and only a moeerate amoung of ester-ester interchange and acidolysis occurred. Diacid glycerides might be prepared from monoglycerides by the procedure which was employed. Saturated diglycerides were esterified with oleic acid with little ester-ester interchange or acidolysis occurring; however, intraesterification was extensive. The reaction between 1,3-distearin and oleic acid yielded approximately 75% 1-oleodistearin and 25% 2-oleodistearin. Saturated diglycerides were esterified with sebacic acid, again with little or no interesterification occurring. Presented at the AOCS meeting in New Orleans, La., 1962; and at the VIth Congress of the International Society for Fat Research, London, England, 1962. A laboratory of the So. Utiliz. Res. and Dev. Div., ARS, U.S.D.A.     

Retarded hydrolysis by pancreatic lipase of seed oils withTrans-3 unsaturation

Triglycerides containing acids withtrans-3 unsaturation (16∶1³, 18∶1³ and 18∶3^{3, 9, 12}) showed a marked retardation of reaction rate with pancreatic lipase. An inverse relationship was found between content oftrans-3 unsaturated acids in seed oils and lipolysis rate. Thetrans-3 unsaturated acids were concentrated in the diglyceride and residual triglyceride fractions of the lipolysates, and only small amounts were present in the free acid and monoglyceride fractions. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Effect of double bond position in octadecenoates upon hydrolysis by pancreatic lipase

Fifteen triacylglycerols containing 12∶0, 14∶0, 16∶0, 18∶2 and one positional isomer ofcis-18∶1 were hydrolyzed by pancreatic lipase (EC 3.1.1.3, glycerol ester hydrolase). The fatty acids in the products of lipolysis were identified and measured by gas liquid chromatography. The substrates containing the Δ2 through Δ7 isomers of 18∶1 were resistant to pancreatic lipolysis. These isomers accumulated in the di- and residual triacylglycerols and were diminished in the free fatty acids. The discrimination was greates against the Δ5 isomer. Scientific Contribution No. 504. Agricultural Experiment Station, University of Connecticut, Storrs, Conn. 06268.     

Uncoupling of catalysis and colipase binding in pancreatic lipase by limited proteolysis

In the intestine, the hydrolysis of triglycerides by pancreaticlipase is performed only in the presence of colipase, whosefunction is to anchor lipase to the bile-salt-coated lipid interface.Biochemical and crystallographk data on porcine and human Upaseshave shown that the molecule is made of two well-delimited domains.In order to get more information on the role of the domainsin catalysis and colipase binding, we performed limited proteolysison lipase from various species and obtained different patternsof cleavage. In the case of porcine and human Upases, only theC-terminal domain (12 kDa) could be obtained after chymotrypticattack, whereas in the horse enzyme the cleavage of the Leu410-Thr411bond gave rise to a large N-terminal (45 kDa) and a small C-terminal(4 kDa) fragment. The isolated porcine and human C-terminaldomains were completely inactive towards emulsified tributyrin,though were able to bind colipase. Conversely, the horse 45kDa fragment retained the lipase activity but failed to correctlybind colipase. This work definitely proves that catalysis andcoUpase binding are separate events involving topographicallydistinct regions of the molecule and focuses attention on therole of the C-terminal domain in colipase binding     

Five-factor response surface optimization of the enzymatic synthesis of citronellyl butyrate by lipase IM77 from Mucor miehei

Response surface methodology (RSM) and a five-level-five-factor central composite rotatable design (CCRD) were used to evaluate the effects of synthetic variables, such as reaction time (3 to 27 h), temperature (25 to 65 °C), enzyme amount (10 to 50%), substrate molar ratio of citronellol to butyric acid (1∶1 to 1∶3), and added water amount (0 to 20%) on molar percent yield of citronellyl butyrate by direct esterification, using lipase IM77 from Mucor miehei. Reaction time and temperature were the most important variables. Substrate molar ratio had no effect on percent molar conversion. Based on contour plots, optimal synthetic conditions were these: reaction time 24 h, temperature 60°C, enzyme amount 20%, substrate molar ratio 1∶1.5, and added water 0%. The predicted molar conversion value was 100%. An actual experimental value of 98% molar conversion was obtained.     

Thermoxidation of substrate models and their behavior during hydrolysis by porcine pancreatic lipase

The behavior of thermoxidized triacylglycerols during hydrolysis catalyzed by porcine pancreatic lipase was evaluated using nonpolar triacylglycerols isolated from palm olein (NPTPO), triolein, and sn-1,3 diolein substrates. Substrates were thermoxidized at 180°C for 1 to 4 h. Owing to formation of polymers and dimers of triacylglycerols, the molecular weight of the thermoxidized substrates increased. After 1 h heating, the concentration of polymers and dimers was similar for the sn-1-3 diolein and triolein samples but higher in NPTPO samples. Conjugated double bonds were formed in all samples, and α,β-unsaturated carbonyl compounds developed through allylic oxidations. These caused increased ultraviolet absorbance at 232 nm. The hydrolysis of heated and unheated samples by the lipase can be described by a Michaelian equation. The enzyme showed a higher apparent V _max and K _M with heated sn-1,3 diolein and triolein than with their unheated counterparts. This was due to the generation of polar compounds which acted as emulsifiers and which favored the formation of an oil/water microemulsion. This behavior was not observed in NPTPO, where heating decreased the apparent V _max and K _M over the first 2 h. Later, a tendency to increase these values was observed. The results could be explained by a balance between concentration of surfactants and of natural emulsifiers in the thermoxidized samples.     

Hydrolysis of intralipid by pancreatic lipase and phospholipase A2-gel filtration studies

Intralipid was incubated with pancreatic lipase (EC 3.1.1.3) and/or phospholipase A₂ (EC 3.1.1.4) at two bile salts/phosphatidylcholine molar ratios and at two different triglyceride hydrolysis rates using various amounts of lipase. Incubations were studied by gel filtration. Results show: (i) During lipase action, three phases of lipids coexist: an emulsified phase, a micellar phase and an intermediate heavy phase sized between the two others. The equilibrium between each phase is dependent upon the bile salts concentration. (ii) Under these conditions, pancreatic lipase was at 60% bound to the emulsified phase, whereas pancreatic phospholipase A₂ was bound at 94% to the micellar phase.