A modified assay system for enzymes involved in N-acetyl group transfer reactions: its use to study enzymes involved in ornithine biosynthesis in plants

An enzymatic method for the determination of the amount of free fatty acids released from triglyceride by lipoprotein lipase is described. The quantity of free fatty acids present in media before and after incubation is measured spectrophotometrically by the oxidation of NADH in the final reaction of a series of coupled enzymatic reactions. This assay for lipoprotein lipase is unlike previously described assays in that radioactive substrates or titration procedures are not used in the free fatty acid determination. In addition, another method for assay of lipoprotein lipase activity that involves the separation of free fatty acids from triglycerides by adsorption chromatography with Florisil as a stationary phase is described.     

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.     

The effects of bovine serum albumin and oleic acid on rat pancreatic lipase and bovine milk lipoprotein lipase

The effects of bovine serum albumin on rat pancreatic lipase and bovine milk lipoprotein lipase were studied in a system of triacylglycerol emulsions stabilized by 1 1 mg/ml albumin. At concentrations greater than 1 mg/ml, albumin inhibited the activity of pancreatic lipase and interfered with enzyme binding to emulsified triacylglycerol particles. These effects could be countered by occupying five fatty acid binding sites on albumin with oleic acid. Following an initial lag period which increased with albumin concentrations, enzyme activity escaped from inhibition presumably due to saturation of fatty acid sites on albumin with oleic acid. Pancreatic lipase was active at 1 mg/ml albumin and 1 mM emulsion-bound oleic acid in the system. The effects of albumin on lipoprotein lipase were diametrically opposed to the above; enzyme activity was completely inhibited by 0.1 mM oleic acid, it increased with increasing fatty acid-free albumin concentrations and decreased as the fatty acid sites on albumin were filled. At 1 mM oleic acid and no added albumin the enzyme failed to bind at the oil water interface, whereas fatty acid-free or saturated albumin had no effect on binding. It is concluded that if the inhibition of pancreatic lipase by albumin is due to the inaccessibility of the enzyme to an oil-water interface blocked by denatured albumin, then albumin saturated with oleic acid would seem to be protected from unfolding at the interface and more readily displaced by the lipase. Pancreatic lipase and lipoprotein lipase, although sharing a number of common features, are distinct enzymes both functionally and mechanistically.     

牡丹品种‘胡红’开花和衰老过程中花瓣脂肪酶活性的变化

以牡丹品种‘胡红’为材料,采用改良铜皂法测定了牡丹花发育过程中花瓣脂肪酶活性的变化。结果表明,牡丹花瓣粗脂肪酶作用的最适pH为6．5,最适温度为35℃。OP乳化剂提高酶活性3．5倍,Mg2＋对酶活性没有影响。露色期（I级）脂肪酶活性和游离脂肪酸含量最高,之后各时期酶活性逐渐下降并维持在低水平;而游离脂肪酸含量在盛开期（V级）有一小高峰。     

假丝酵母Candida rugosa产脂肪酶条件的优化

对假丝酵母Candidarugosa产脂肪酶的条件进行了优化.比较实验证明,碳源是影响酶产量的主要因素.其中,糖类使细胞生长良好,但酶产量较低,而脂类是较为适合的碳源.本实验首次发现长链的不饱和脂肪酸酯,三油酸甘油酯是最好的碳源.氮源的影响较小.而添加物的使用是有效提高脂肪酶产量的另一种方法.PVA可促进碳源的乳化,从而提高脂肪酶产量.吐温虽没有促进细胞生长,但却大幅度提高了酶的产量.将以上优化的结果应用于发酵罐中,分批流加操作时,脂肪酶产量高达每毫升128.2单位,为目前报道的最高值.     

Synthesis activity-based zymography for detection of lipases and esterases

A new zymography method for lipases and esterases was developed on the basis of the esterification reaction between fatty acids and alcohols. The enzymes were separated by SDS-PAGE and native PAGE. The gel was washed and then incubated in an aqueous solution containing fatty acids (oleic acid 18:1 or caprylic acid 8:0) and dodecanol. Synthesis was visualized by in situ precipitation of water-insoluble and non-diffusible fatty acid esters, such as dodecyl oleate and dodecyl octanoate. The synthesis activity-based zymography was confirmed with different enzyme samples, including commercial lipase preparations, purified recombinant lipase and cutinase, and crude culture supernatants of lipolytic enzyme-producing soil bacteria.     

Diacylglycerol lipase and kinase activities in rat brain microvessels

Diacylglycerols can accumulate transiently in intact cells as a consequence of the degradation of phosphatidylinositol by phospholipase C, but little information is available concerning their metabolic fate in the vascular endothelium. Diacylglycerol lipase and kinase activities were measured in rat brain microvessel preparations. Lipase activity, measured by the release of free fatty acids, was much greater at pH 4.5 than at pH 7. The acid lipase was predominantly particulate and likely originated in lysosomes, whereas the neutral lipase was mainly soluble. The fatty acid at the sn-1 position of the diacylglycerol substrate was hydrolyzed faster than that at the sn-2 position at both pH 4.5 and 7. The 2-monoacylglycerol accumulated at pH 4.5 but not at 7 due to the presence of a monoacylglycerol lipase activity with a neutral pH optimum. The formation of phosphatidic acid (kinase activity) was also measured in microvessels. When lipase and kinase activities were measured simultaneously, the formation of phosphatidic acid from a 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol substrate was 4-fold greater than the release of fatty acid (oleate) from the sn-2 position. Introduction of arachidonic acid to the sn-2 position of the diacylglycerol substrate increased kinase activity but reduced lipase activity. The release of fatty acids from the sn-2 position of phosphatidic acid could not be detected.     

Effects of Fatty Acids,Lipase Activator,Phospholipids and Related Substances on the Lipase Production by Candida paralipolytica

Unsaturated long-chain fatty acids such as oleic, linoleic and ricinoleic acid except for elaidic acid were effective on the lipase production by Candida paralipolytica, but saturated fatty acids were not effective. When elaidic and myristic acids were dissolved in n-hexadecane to be dispersed in a liquid state, they became to be effective on the lipase production. These results suggest that long-chain fatty acids of a liquid state are effective on the lipase production. Lipase activator A and phosphatidylethanolamine stimulated the effect of fatty acids on the lipase production. Effects of sterols and surface-active substances on the lipase production were also reported.

A weak tributyrin-hydrolyzing activity, in the absence of fatty acids, was found in the yeast cell.     

pH as an indicator of free fatty acid release from adipocytes.

A convenient method of measuring initial rates of free fatty acid efflux from isolated adipocytes during triglyceride breakdown by hormone-sensitive lipase is described. The procedure is based on the dissociation of protons from carboxyl groups of free fatty acids. A recording pH meter is used to monitor H+ concentration in the medium continuously as an index of free fatty acid release. A stoichiometric relationship was demonstrated between proton release and extracellular free fatty acid concentration as determined by the 63Ni radioassay method of Ho (1970. Anal. Biochem. 36: 105-113). An acid pH (6.8) caused a reduction in proton release, which was immediately and completely reversed by raising the pH to 7.4.     

Properties of triacylglycerol lipase in a mitochondrial fraction from baker's yeast (Saccharomyces cerevisiae).

A triacylglycerol lipase in a mitochondrial fraction isolated from yeast (Saccharomyces cerevisiae) has been characterized and the hydrolysis studied kinetically using an insoluble artificial triacylglycerol suspension. 1. The triacylglycerol was hydrolyzed almost completely to fatty acids and glycerol. The lipase activity was inhibited by potassium fluoride and the sodium salts of -chloride, -glycocholate and -pyrophosphate as well as by protamine sulfate but at concentrations much too high to indicate that the lipase is a non specific esterase or a lipoprotein lipase. Also parachloromercuribenzoate inhibited the lipase activity. Inhibitory effect of fatty acid was observed at concentrations above 1mM. This inhibition may provide a regulatory mechanism of the lipase in vivo. 2. On the day of isolation the lipase activity of intact mitochondria at pH 7.5 and 30 degrees C was 400 nmol free fatty acid -h-1 - mg-1 at a triacylglycerol concentration of 9.0 mM. Sonication of the mitochondria increased the activity 2-3 fold. Freezing of the mitochondria also activated the lipase and this activation was dependent upon the freezing method, the concentration of mitochondrial protein and the presence of bovine serum albumin. 3. The particulate nature of the assay system was illustrated by the observation that the apparent Km value of the lipase increased with the concentration of mitochondrial protein. For each protein concentration the lipase had two apparent Km values when the activity was assayed with intact mitochondria, but only one when assayed with submitochondrial particles. At the same protein concentration the Km value for the latter was identical with the "low affinity" Km for the lipase in intact mitochondria.     

An ultraviolet spectrophotometric assay for measuring lipase activity using long-chain triacyglycerols from Aleurites fordii seeds

In this study, we designed a specific, continuous, and sensitive UV spectrophotometric lipase assay using natural triacylglycerols (TAGs) from the Aleurites fordii seed oil (tung oil). alpha-Eleostearic acid (9,11,13-cis, trans,trans-octadecatrienoic acid) is the main fatty acid component (it accounts for up to 70%) of the TAGs from tung oil. The conjugated triene present in alpha-eleostearic acid constitutes an intrinsic chromophore, which confers strong UV absorption properties on both the free fatty acid and the TAGs from tung oil. The lipase assay is based on the difference between the apparent molar extinction coefficients of the two types of alpha-eleostearic acid present, that which is esterified into TAGs and that which is released into the reaction medium. This difference is responsible for the variations in the UV absorption spectrum of the reaction medium occurring upon enzymatic TAGs hydrolysis. Using the purified lipase from Thermomyces lanuginosa (TLL) and the detergent sodium taurodeoxycholate (NaTDC, 4 mM), it was established that the most suitable method of measuring lipolysis consisted of monitoring the decrease in the OD at 292 nm, which was linear with time and proportional to the amount of lipase added. In order to be able to estimate the specific activity of TLL, we determined an apparent molar extinction coefficient of alpha-eleostearic acid (epsilon = 13,900 M(-1) cm(-1)) under the assay conditions. Amounts of pure TLL as small as 1 ng can be easily detected in the presence of 4 mM NaTDC. Interestingly, the NaTDC concentration can be decreased as far as 0.05 mM. In comparison with other well-known methods of lipase assay, the detection limit of this new method is 100-fold lower than with the pH-stat method and similar to that of a fluorescent assay recently developed at our laboratory.     

Effect of phosphatidylcholine and free fatty acids on the activity of pancreatic lipase-colipase

Mixed micelles of bile salt and phospholipids inhibit the lipase-colipase-catalysed hydrolysis of triacylglycerols. Free fatty acids can reverse this inhibition and reactivate lipase-colipase. This reactivation is either due to the formation of a high-affinity complex between lipase and colipase induced by free fatty acids and/or to a change of the quality of the interface. Lauric acid, oleic acid and linoleic acid are the most potent reactivators, while short-chain free fatty acids have no effect and long-chain, saturated free fatty acids inhibit the lipase-colipase activity further. The physiological relevance of these results is evident as the glyceride emulsion reaching the duodenum already contains free fatty acids due to the activity of lingual lipase in the stomach.     

Synthesis of glycerides containing n-3 fatty acids and conjugated linoleic acid by solvent-free acidolysis of fish oil

Menhaden oil, a rich source of n-3 fatty acids, was interesterified with conjugated linoleic acid (CLA) in a reaction medium composed solely of substrates and either free or immobilized commercial lipase preparations. Of five lipases tested, an immobilized preparation from Mucor miehei provided the fastest rate of incorporation of CLA into fish oil acylglycerols; however, and as observed with most of the lipases utilized, a significant proportion of the n-3 fatty acid residues were liberated in the process. A soluble lipase from Candida rugosa converted free CLA to acylglycerol residues while leaving the n-3 fatty acid residues virtually untouched. Even though the reaction rate was slower for this enzyme than for the other four lipase preparations, the specificity of the free C. rugosa lipase gives it the greatest potential for commercial use in preparing fish oils enriched in CLA residues but still retaining their original n-3 fatty acid residues.     

One-pot synthesis of polyunsaturated fatty acid amides with anti-proliferative properties

A one-pot environmentally friendly transamidation of ω-3 fatty acid ethyl esters to amides and mono- or diacylglycerols was investigated via the use of a polymer-supported lipase. The method was used to synthesize a library of fatty acid monoglyceryl esters and amides. These new derivatives were found to have potent growth inhibition effects against A549 lung cancer cells.     

Hydrolysis of chylomicron polyenoic fatty acid esters with lipoprotein lipase and hepatic lipase

The lipolysis of rat chylomicron polyenoic fatty acid esters with bovine milk lipoprotein lipase and human hepatic lipase was examined in vitro. Chylomicrons obtained after feeding fish oil or soy bean oil emulsions were used as substrates. The lipolysis was followed by gas chromatography or by using chylomicrons containing radioactive fatty acids. Lipoprotein lipase hydrolyzed eicosapentaenoic (20:5) and arachidonic acid (20:4) esters at a slower rate than the C14-C18 acid esters. More 20:5 and 20:4 thus accumulated in remaining tri- and diacylglycerols. Eicosatrienoic, docosatrienoic and docosahexanoic acids exhibited an intermediate lipolysis pattern. When added together with lipoprotein lipase, hepatic lipase increased the rate of lipolysis of 20:5 and 20:4 esters of both tri- and diacylglycerols. Addition of NaCl (final concentration 1 M) during the course of lipolysis inhibited lipoprotein lipase as well as the enhancing effect of hepatic lipase on triacylglycerol lipolysis. Hepatic lipase however, hydrolyzed diacylglycerol that had already been formed. Chylomicron 20:4 and 20:5 esters thus exhibit a relative resistance to lipoprotein lipase. It is suggested that the tri- and diacylglycerol species containing these fatty acids may accumulate at the surface of the remnant particles and act as substrate for hepatic lipase during a concerted action of this enzyme and lipoprotein lipase.     

Effect of rearing method on the storage of fats by adipose tissue in sheep

Adipocyte synthesis de novo and lipoprotein lipase activity have been used simultaneously to measure the lipogenic activity of adipose tissue in sheep. Acetate and glucose were used as precursors of fatty acid synthesis. The sheep were raised either outdoors or in a sheepfold. They were slaughtered by lots at mean weights of 24 and 32.5 kg. Compared to lipoprotein lipase activity, de novo synthesis of fatty acids was the main way of constituting lipid depositions. Raising the sheep outdoors favored the use of glucose as precursor of lipid synthesis at the first slaughter stage at 24 kg. Later at 32.5 kg, glucose utilization was practically zero compared to acetate, whatever the mode of rearing. The NADPH production needed for fatty acid synthesis was almost entirely due to NADP isocitrate dehydrogenase activity. Variations in both de novo synthesis and in lipoprotein lipase activity in relation with rearing method and slaughter weight were especially evident in the group raised outdoors.     

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.     

Isolation and functional expression of a novel lipase gene isolated directly from oil-contaminated soil

A lipase gene SR1 encoding an extracellular lipase was isolated from oil-contaminated soil and expressed in Escherichia coli. The gene contained a 1845-bp reading frame and encoded a 615-amino-acid lipase protein. The mature part of the lipase was expressed with an N-terminal histidine tag in E. coli BL21, purified and characterized biochemically. The results showed that the purified lipase combines the properties of Pseudomonas chlororaphis and other Serratia lipases characterized so far. Its optimum pH and temperature for hydrolysis activity was pH 5.5-8.0 and 37°C respectively. The enzyme showed high preference for short chain substrates (556.3±2.8 U/μg for C10 fatty acid oil) and surprisingly it also displayed high activity for long-chain fatty acid. The deduced lipase SR1 protein is probably from Serratia, and is organized as a prepro-protein and belongs to the GXSXG lipase family.     

Substrate Specificity and Mode of Action of the Lipase of Thermophilic Fungus Humicola lanuginosa S-38

Substrate specificity of the lipase of thermophilic fungus, Humicola lanuginosa S–38, was investigated. It was found that the lipolytic activity was greatly influenced by the structure of both fatty acid and alcohol moieties of the substrate. It was concluded that the hydrolysis of both water soluble and water insoluble ester was catalyzed by the Humicola lipase itself. The Humicola lipase showed no positional specificity and split ester bonds on all positions of triolein at about the same rate. Both palmitic acid (α) and linoleic acid (β) ester bonds of phosphatidyl-ethanolamine were split indicating no positional specificity of fatty acid ester bonds. From above results, it was made clear that mode of action of Humicola lipase on triolein and on phosphatidyl-ethanolamine is identical. The Humicola lipase had no activity of lipoprotein lipase.     

On the performance of a hollow-fiber bioreactor for acidolysis catalyzed by immobilized lipase

The present communication describes the chemical modification of anhydrous butterfat by interesterification with oleic acid catalyzed by a lipase of Mucor javanicus. Two reactor configurations were tested, a batch-stirred tank reactor containing suspended lipase and a batch-stirred tank reactor in combination with a hollow-fiber membrane module containing adsorbed lipase. The goal of this research was to assess the advantage of using a (hydrophobic) porous support to immobilize the lipase in attempts to engineer butterfat with increased levels of unsaturated fatty acid residues (oleic acid) at the expense of medium-to-long chain saturated fatty acids (myristic and palmitic acids). Reactions were carried out at 40 degrees C in the absence of solvent under controlled water activity, and were monitored by chromatographic assays for free fatty acids. The results obtained indicate that the rate of interesterification using the proposed reactor configuration is enhanced by a factor above 100 relative to that using suspended lipase, for the same protein mass basis. Although hydrolysis of butterfat occurred to some degree, the enzymatic process that uses the hollow-fiber reactor was technically superior to the stirred tank system. Copyright 1998 John Wiley & Sons, Inc.