Hydrolysis of fully esterified alcohols containing from one to eight hydroxyl groups by the lipolytic enzymes of rat pancreatic juice

The enzymatic hydrolysis in vitro of the esters of methanol, ethylene glycol, glycerol, erythritol, pentaerythritol, adonitol, sorbitol, and sucrose in which all alcohol groups were esterified with oleic acid was studied. Various preparations of rat pancreatic juice, including pure lipase, were used as the sources of enzymes. Lipase (EC 3.1.1.3) did not hydrolyze compounds that contained more than three ester groups. Compounds containing four and five ester groups were hydrolyzed by certain preparations of pancreatic juice; this activity is attributed to the enzyme, nonspecific lipase. This enzyme also hydrolyzed esters of primary alcohols. The compounds containing six (sorbitol) and eight (sucrose) ester groups were not hydrolyzed.     

Substrate specificities of lipases from corn and other seeds

Lipases from several seed species were shown to be relatively specific on triacylglycerols containing the major fatty acid components of the storage triacylglycerols in the same species. In a direct comparison using individual triacylglycerol as well as mixed triacylglycerol preparations, highest activities were observed in corn lipase on trilinolein and triolein, castor bean lipase on triricinolein, rapeseed lipase on trierucin, and elm seed lipase on tricaprin. This pattern of fatty acyl specificity was also observed on diacylglycerols, monoacylglycerols, and fatty acyl 4-methylumbelliferone, although the pattern became less distinct. The seed lipases were inactive on lecithins. Corn lipase was more active on tri- than di- or monolinolein, and released linoleic acids from both primary and secondary positions. As judged from the kinetics of hydrolysis of rac-glyceryl-2,3-stearate-1-oleate and rac-glyceryl-1,3-stearate-2-oleate, and of trilinolein and dilinoleins, corn lipase exerted some degree of preference in releasing fatty acid from the primary than the secondary position of a triacylglycerol. At the primary position, corn lipase was more active on oleyl ester than stearyl ester.     

Carboxylic ester hydrolases of rat pancreatic juice

An attempt was made to establish the number and characteristics of the enzymes in pancreatic juice that hydrolyze nitrogen- and phosphorus-free esters of fatty acids. For this purpose model compounds were hydrolyzed by lyophilized rat pancreatic juice under conditions that accelerated or inhibited the reactions. Although it is not established with certainty, it is suggested that three enzymes are responsible for the hydrolysis of fatty acid esters. The first enzyme is glycerol-ester hydrolase (EC 3.1.1.3) or lipase. This enzyme hydrolyzes water-insoluble esters of primary alcohols. The reaction occurs at an oil/water interface and is inhibited by bile salts at pH 8. The enzyme is relatively stable at pH 9, but unstable at pH 4. It has a broad pH optimum between 7.5 and 9.5. The second enzyme hydrolyzes esters of secondary alcohols and of other alcohols as well. It has an absolute requirement for bile salts and has a pH optimum at about 8. The enzyme is unstable in pancreatic juice when maintained at pH 9, probably due to the action of trypsin. It may be identical with sterol-ester hydrolase (EC 3.1.1.13). The third enzyme hydrolyzes water-soluble esters. It too has an absolute requirement for bile salts, although a smaller amount is necessary for maximum activity. This enzyme also is unstable at pH 9, but can be differentiated from the preceding enzyme by its stability at pH 4 and its pH optimum of 9.0. Carboxylic-ester hydrolase (EC 3.1.1.1) is not found in pancreatic juice, although it is present in pancreatic tissue.     

Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol, medium chain glycerides and PEG esters

Labrasol is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive lipases. The hydrolysis of Labrasol by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol. These lipases were found to hydrolyze the main components of Labrasol (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol(R) is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol taken orally.     

Glyceride Structure and Biosynthesis of Natural Fats

In order to study specificity of pancreatic lipase, a number of synthetic triglycerides were hydrolyzed by the enzyme under an improved condition. The proportions of isomers of the derived mono- and diglycerides, and the fatty acid compositions of the derived free acids and monoglycerides were determined. The hydrolyzing rate of fatty acids in glycerides depended on the position esterified in the glycerol, carbon number of the acid, and structure of the glyceride. Positional specificity of the enzyme was markedly displayed for symmetrical triglyceridcs composed of long chain acids, but at somewhat lower rate for glycerides containing short chain or highly unsaturated acids.     

Acid Carboxypeptidase-like Activity Contaminating Proctase B

Ester synthesis by the purified lipase from Pseudomonas fragi 22.39 B was investigated. The lipase could synthesize esters from oleic acid and primary or secondary alcohols, but it did not react with tertiary alcohols. Also, the enzyme could use the fatty acids with straight carbon chains as substrates. The activity was enhanced by increasing the carbon number of the fatty acid, but this is not the case for alcohol. The lipase synthesized glycerides from glycerol and oleic acid. 1(3)-Monoolein and 1,3-diolein were the main products and triolein was minor. Synthesis of monoester such as butyl oleate was scarcely affected by the water content in the reaction mixture, while that of glyceride of oleic acid was much affected.     

Digestion in vitro of erythritol esters by rat pancreatic juice enzymes

The mechanism of the digestion of erythritol esters was determined using rat pancreatic juice and purified pancreatic lipase (EC 3.1.1.3). Conditions of hydrolysis were used that would selectively activate or inactivate nonspecific lipase or lipase. It was shown that erythritol tetraoleate was hydrolyzed by nonspecific lipase but not by lipase. The initial digestion product was a triester, predominantly erythritol-1,2,3-trioleate. Thus, nonspecific lipase preferentially hydrolyzed the ester of a primary alcohol. In contrast to the results obtained with the tetraester, lipase could remove a fatty acid from the triester but the resulting erythritol-2,3-dioleate was not hydrolyzed by lipase. The selectivity of this hydrolysis and the inability to hydrolyze the diester are attributed to the known specificity of this enzyme to act only on esters of primary alcohols. Nonspecific lipase completely hydrolyzed erythritol tetraoleate to free erythritol in a stepwise manner. The relative rates of these reactions were tetraester --> triester --> diester --> monoester --> erythritol Because of the specificity of pancreatic lipase and the lack of specificity of nonspecific lipase it is likely that this latter enzyme is the primary agent for the hydrolysis of erythritol esters in the intact animal.     

Glyceride synthesis by four kinds of microbial lipase

Apart from their usual mechanism of action, lipases from Aspergillus niger and Rhizopus delemar also catalyzed the synthesis of glycerides from oleic acid and glycerol. Lipases from Geotrichum candidum and Penicillium cyclopium were inactivated by oleic acid, but were stable in the presence of casein, albumin or buffer of appropriate pH. Lipases from Aspergillus niger and Rhizopus delemar synthesized glycerides from, not only fatty acid, but dibasic acids and aromatic acids, making ester bonds only at position 1 and 3 of glycerol. In contrast, lipases from Geotricum candidum and Penicillium cyclopium synthesized glycerides only from long chain fatty acids, and made ester bonds at all three available positions of the glycerol molecule.     

Fatty acids generated by gastric lipase promote human milk triacylglycerol digestion by pancreatic colipase-dependent lipase

The concerted action of purified bovine gastric lipase and human pancreatic colipase-dependent lipase and colipase, or crude human pancreatic juice, in the digestion of human milk triacylglycerols was explored in vitro. Gastric lipase hydrolyzed milk triacylglycerol with an initially high rate but became severely inhibited already at low concentration of released fatty acid. In contrast, colipase-dependent lipase could not, by itself, hydrolyze milk triacylglycerol. However, a short preincubation of milk with gastric lipase, resulting in a limited lipolysis, made the milk fat triacylglycerol available for an immediate and rapid hydrolysis by pancreatic juice, and also for purified colipase-dependent lipase, provided colipase and bile salts were present. The same effect was obtained when incubation with gastric lipase was replaced by addition of long-chain fatty acid. Long-chain fatty acid increased the binding of colipase-dependent lipase to the milk fat globule. Binding was efficient only in the presence of both fatty acid and colipase. We conclude that a limited gastric lipolysis of human milk triacylglycerol, resulting in a release of a low concentration of long-chain fatty acids, is of major importance for the subsequent hydrolysis by colipase-dependent lipase in the duodenum.     

Kinetic properties of turkey pancreatic lipase: a comparative study with emulsified tributyrin and monomolecular dicaprin

Using the classical emulsified system and the monomolecular film technique, we compared several interfacial properties of turkey pancreatic lipase (TPL) and human pancreatic lipase (HPL). TPL, like HPL, presented the interfacial activation phenomenon when vinyl ester was used as substrate. In the absence of colipase and bile salts, using tributyrin emulsion or monomolecular films of dicaprin at low surface pressure, TPL, unlike HPL, hydrolyzes pure tributyrin emulsion as well as dicaprin films maintained at low surface pressures. TPL was also able to hydrolyze triolein emulsion in the absence of any additive and despite the accumulation of long-chain free fatty acids at the interface. The difference of behaviors between TPL and HPL can be explained by the penetration power of each enzyme. The enzyme that presents the maximal pi(c) (TPL) interacts more efficiently with interfaces, and it is not denaturated at high interfacial energy. Turkey pancreatic lipase is more active on rac-dicaprin than HPL; a maximal ratio of 9 was found between the catalytic activities of the two lipases measured at their surface pressure optima (20 mN m(-1)). A kinetic study on the surface pressure dependency, stereospecificity, and regioselectivity of TPL was performed using enantiopure diglyceride (1,2-sn-dicaprin and 2,3-sn-dicaprin) and a prochiral isomer (1,3-dicaprin) that were spread as monomolecular films at the air-water interface. At low surface pressure (15 mN m(-1)), TPL acts preferentially on primary carboxylic ester groups of the diglyceride isomers (1,3-dicaprin), but at high surface pressure (23 mN m(-1)), this enzyme prefers both adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin). HPL prefers adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin). Furthermore, TPL was found to be markedly stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin at low surface pressure (15 mN m(-1)), while at high surface pressure (23 mN m(-1)), this lipase presents a stereopreference for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. HPL is stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin both at 15 and 23 mN m(-1).     

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).     

Purification and substrate specificity of Staphylococcus hyicus lipase

The Staphylococcus hyicus lipase gene has been cloned and expressed in Staphylococcus carnosus. From the latter organism the enzyme was secreted into the medium as a protein with an apparent molecular mass of 86 kDa. This protein was purified, and the amino-terminal sequence showed that the primary gene product was indeed cleaved at the proposed signal peptide cleavage site. The protein was purified from large-scale preparations after tryptic digestion. This limited proteolysis reduced the molecular mass to 46 kDa and increased the specific activity about 3-fold. Although the enzyme had a low specific activity in the absence of divalent cations, the activity increased about 40-fold in the presence of Sr2+ or Ca2+ ions. The purified lipase has a broad substrate specificity. The acyl chains were removed from the primary and secondary positions of natural neutral glycerides and from a variety of synthetic glyceride analogues. Thus triglycerides were fully hydrolyzed to free fatty acid and glycerol. The enzyme hydrolyzed naturally occurring phosphatidylcholines, their synthetic short-chain analogues, and lysophospholipids to free fatty acids and water-soluble products. The enzyme had a 2-fold higher activity on micelles of short-chain D-lecithins than on micelles composed of the L-isomers. Thus the enzyme from S. hyicus has lipase activity and also high phospholipase A and lysophospholipase activity.     

The activities of lipases and carnitine palmitoyl-transferase in muscles from vertebrates and invertebrates

1. The activities of tri-, di- and mono-glyceride lipase and carnitine palmitoyltransferase were measured in homogenates of a variety of muscles. These activities were used to estimate the rate of utilization of glycerides and fatty acids by muscle. In muscles whose estimated rates of fat utilization can be compared with rates calculated for the intact muscle from such information as O₂ uptake, there is reasonable agreement between the estimated and calculated rates. 2. In all muscles investigated the maximum rates of hydrolysis of glycerides increase in the order triglyceride, diglyceride, monoglyceride. The activity of diglyceride lipase is highest in the flight muscles of insects such as the locust, waterbug and some moths and is lowest in the flight muscles of flies, bees and the wasp. These results are consistent with the utilization of diglyceride as a fuel for some insect flight muscles. 3. In many muscles from both vertebrates and invertebrates the activity of glycerol kinase is similar to that of lipase. It is concluded that in these muscles the metabolic role of glycerol kinase is the removal of glycerol produced during lipolysis. However, in some insect flight muscles the activity of glycerol kinase is much greater than that of lipase, which suggests a different role for glycerol kinase in these muscles.     

利用麻风树油驯化筛选高活性脂肪酶产生菌及其催化酯化反应

以1株分解麻风树油的脂肪酶产生菌Pseudomonas sp. LP-1为出发菌株, 通过麻疯树油定向驯化筛选获得1株酶活较高且产酶稳定的菌株P. sp. X-2-45, 其水解酶活为29.79 U/mL, 比原始菌株提高了288%。对P. sp. X-2-45生长与产酶特征、对植物油脂水解能力及在有机相中催化脂肪酸和有机醇间的酯化反应研究发现, 该菌株生长速率和产酶速率明显加快, 培养30 h时生物量和酶活达到最大, 稳定期延长, 培养过程中脂肪酶在培养基中的稳定性提高。以麻疯树油诱导合成的P. sp. X-2-45脂肪酶对麻疯树油的水解能力比原始菌株提高了378%, 说明采用麻风树油定向驯化可提高脂肪酶对相应底物的水解能力。X-2-45脂肪酶可以催化月桂酸与正丁醇、正辛醇、月桂醇和丙三醇之间, 棕榈酸、硬脂酸与甲醇、正辛醇、月桂醇和丙三醇之间, 油酸与甲醇、正丁醇、正辛醇、月桂醇和丙三醇之间发生酯化反应。     

Characteristics of immobilized lipase on hydrophobic superparamagnetic microspheres to catalyze esterification

A novel immobilized lipase (from Candida rugosa) on hydrophobic and superparamagnetic microspheres was prepared and used as a biocatalyst to catalyze esterification reactions in diverse solvents and reaction systems. The results showed that the immobilized lipase had over 2-fold higher activities in higher log P value solvents. An exponential increase of lipase activity against log P of two miscible solvent mixtures was observed for the first time. Both free and immobilized lipase achieved its maximum activity at the range of water activity (a(w)) 0.5-0.8 or higher. At a(w) 0.6, the immobilized lipase exhibited markedly higher activities in heptane and a solvent-free system than did the native lipase. In multicompetitive reactions, the alcohol specificity of the lipase showed a strong chain-length dependency, and the immobilized enzyme exhibited more preference for a longer-chain alcohol, which is different from previous reports. The immobilized lipase showed higher specificities for butyric acid and the medium-chain-length fatty acids (C(8)-C(12)). Then, the immobilized lipase was extended to solvent-free synthesis of glycerides from glycerol and fatty acids. Recovered by magnetic separation, the immobilized lipase exhibited good reusability in repeated batch reaction, indicating its promising feature for biotechnology application.     

Subcellular fractionation, partial purification and characterization of neutral triacylglycerol lipase from pig liver.

The subcellular distributions of acidic (pH 4.5) and neutral (pH 7.5) longchain triacylglycerol lipases (glycerol ester hydrolase, EC 3.1.1.3) of pig liver have been determined. The distribution of the acidic lipase closely paralleled that of the lysosomal marker enzyme, cathepsin D. Approx. 60% of the neutral lipolytic activity resided in the soluble fraction;the distribution of this activity failed to parallel that of marker enzymes for mitochondria, lysosomes, microsomes, or plasma membranes. A method has been developed for purification of the neutral lipase from the soluble fraction by ultracentrifugation. An approximate 90-fold purification was achieved, with recovery of 16% of the initial activity. The partially purified neutral lipase exhibited a pH optimum between 7.25 and 7.5. It required 30 mM emulsified triolein for optimal activity and ceased to liberate fatty acids after 30 min of incubation. The enzymatic activity was destroyed by heating at 60 degrees C. Neutral lipase was inhibited by sodium deoxycholate, Triton X-100 and iodoacetamide. The activity was not inhibited by sodium taurocholate, EDTA, heparin and diethyl-p-nitrophenyl phosphate. Neutral lipase failed to exhibit activity in assay systems specific for lipoprotein lipase, monoolein hydrolase, tributyrinase, and methyl butyrate esterase and showed little or no capacity to hydrolyze chyle chylomicrons or plasma very low density lipoproteins. It is suggested that the function of neutral lipase may be to supply the liver with fatty acids liberated from endogenously synthesized or stored triacylglycerols.     

THE INCORPORATION OF [1-14C]ACETATE INTO UNESTERIFIED FATTY ACIDS IN RAT CEREBRAL CORTEX IN VIVO

—The incorporation of [1-¹⁴C]acetate into unesterified fatty acids and into the fatty acids of neutral glycerides and of phospholipids has been measured in rat cerebral cortex in vivo. The most rapid incorporation is seen in the unesterified fatty acids which have a turnover time of 5-6 min. It is suggested that unesterified fatty acids are precursors to neutral glycerides and phospholipids rather than being derived from them by lipase activity.     

Purification of pancreatic carboxylic-ester hydrolase by immunoaffinity and its application to the human bile-salt-stimulated lipase

A column of immobilized antibodies directed against pure human pancreatic carboxylic (cholesterol) ester hydrolase was used to purify in a single step the enzyme from human pancreatic juice as well as carboxylic-ester hydrolases from other species (rat, dog). This immunoaffinity method was also used for the purification of the related bile-salt-stimulated lipase from the human skim milk. The enzymes were homogeneous on SDS-PAGE. The yields obtained were always higher than those previously observed using either conventional or affinity columns. The human and dog carboxylic-ester hydrolases as well as the bile-salt-stimulated lipase, in contrast to the rat enzyme, are glycoproteins. From our results, it can be speculated that these enzymes, which differ in their molecular weight but not in their N-terminal sequences or amino-acid compositions, might have a similar proteic core with a molecular mass between 65 and 75 kDa. The difference in their respective molecular masses might result from a different level of glycosylation of pancreatic carboxylic-ester hydrolases (and milk bile-salt-stimulated lipase).     

Depolymerization of a hydroxy fatty acid biopolymer, cutin, by an extracellular enzyme from Fusarium solani f. pisi: isolation and some properties of the enzyme

Fusarium solani f. pisi was shown to grow on the hydroxy fatty acid biopolymer cutin as the sole carbon source. Such growth conditions induced the production of an extracellular cutin depolymerising enzyme. Analysis of products enzymatically derived from labeled cutin by thin-layer chromatography and radio gas-liquid chromatography showed that the Fusarium enzyme released all classes of cutin monomers. This enzyme preparation also catalyzed hydrolysis of several model ester substrates. It did not hydrolyze triacyl glycerol and pancreatic lipase did not hydrolyze cutin, indicating that the Fusarium enzyme is not a nonspecific lipase. With p-nitrophenyl palmitate as the model substrate the enzyme showed a broad pH optimum near 8.5 and it was stimulated by Triton X-100. Maximal stimulation was obtained at 3.7 mg/ ml of the detergent. Apparent K_m for p-nitrophenyl palmitate was 1.6 × 10^?4m. p-Nitrophenyl esters of C₂–C₁₈ acids gave comparable values for K_m and V revealing no striking specificity. Treatment with diisopropyl fluorophosphate severely inhibited the enzyme while iodoacetamide and p-chloromercuric benzoate did not affect the enzymatic activity, suggesting that the Fusarium enzyme is a serine hydrolase.     

Characteristics of immobilized lipase-catalyzed hydrolysis of olive oil of high concentration in reverse phase system

Candida rugosa lipase immobilized by adsorption on swollen Sephadex LH-20 could almost completely hydrolyze 60% (v/v) olive oil in isooctane. Kinetic analysis of the lipase-catalyzed hydrolysis reaction was found to be possible in this system. Amount of fatty acids produced was linearly proportional to the enzyme concentration of 720 mug/g wet gel. The specific enzyme activity was 217 units/mg protein at 60% (v/v) olive oil concentration. When the initial rate is plotted versus concentration of olive oil, this system did not follow Michaelis-Menten kinetics. Maximum activity was obtained at pH 7, but optimum temperature shifted towards higher one with the increase of olive oil concentration. Among the various chemical compounds tested, Hg(2+) and Fe(2+) inhibited the lipase seriously. As the concentration of olive oil increased, the rate of the hydrolysis also increased, but degree of the hydrolysis was observed to decrease. The supply of water from the inside of the gel to the surface of the gel was the main factor for the control of the rate of hydrolysis in batch hydrolysis. The immobilized lipase was used to hydrolyze olive oil two times. Achievement of chemical equilibrium took a longer time with the addition of water and the degree of hydrolysis decreased in the second consecutive trial. After the second hydrolysis trial, the gels were regenerated in a packed column first by eluting out both residual fatty acids around the gel particles and the accumulated glycerol with ethanol and then with 0.05M phosphate buffer, pH 7. The immobilized lipase on the regenerated gel showed the same hydrolysis activity as the original one.