Characterization of lipases from the lipid bodies and microsomal membranes of erucic acid-free oilseed-rape (Brassica napus) cotyledons.

Lipase (triacylglycerol lipase, EC 3.1.1.3) activities have been reported previously in the lipid body and microsomal membranes of oilseed-rape (Brassica napus cv. Andor) seedlings, but conflicting data made it unclear whether there was one lipase in the lipid bodies, with the microsomal activity being attributable to fragments of lipid-body membrane, or if there were two separate lipase activities. In the present study, simultaneous characterization of the lipases under identical conditions showed they differed substantially in their pH-activity curves, kinetics and substrate specificities. (1) The kinetics of the microsomal lipase showed that the rate of lipolysis reached a plateau at concentrations above 5 mM, whereas the lipid-body lipase showed a linear increase in activity with substrate concentration up to 20 mM. (2) The pH optimum of the microsomal lipase was 7.5, whereas that of the lipid-body lipase was 9.0. The microsomal lipase was greatly inhibited at higher pH values, whereas the lipid-body lipase was much less affected. (3) Activity of the microsomal lipase was greatly diminished when substrates with longer chain length were used, and enhanced 4-fold if the substrates contained a single double bond. The lipid-body lipase was relatively unaffected by the type of fatty acid in the triacylglycerol. (4) SDS/polyacrylamide-gel electrophoresis showed little or no cross-contamination of the lipid-body and microsomal fractions. (5) The microsomal lipase activity comprised 75-80% of the total extracted.     

Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.     

Purification and characterization of hepatic lipase from Todarodes pacificus

Lipase was purified from squid (Todarodes pacificus) liver in an attempt to investigate the possibility of applying the enzyme for biotechnological applications. Crude extract of squid liver was initially fractionated by the batch type ion exchange chromatography. The fraction containing lipase activity was further purified with an octyl-Sepharose column. Finally, lipase was purified by eluting active protein from a non-dissociating polyacrylamide gel after zymographic analysis. Molecular weight of the purified enzyme was determined to be 27 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity at a temperature range of 35-40 degrees C and at pH 8.0. The activity was almost completely inhibited at 1 mM concentration of Hg(2+) or Cu(2+) ion. Partial amino acid sequence of the enzyme was also determined.     

Separation and characterization of two molecular forms of Geotrichum candidum lipase

Southern blot analysis of the Geotrichum candidum genome with a cloned lipase cDNA as the probe indicated the existence of two genes on the chromosome of the fungus which are homologous to the cDNA. As expected, two forms of lipase (lipases I and II) were actually isolated by hydrophobic interaction chromatography after a multistep procedure including ammonium sulfate fractionation, anion exchange chromatography, and gel filtration of the culture filtrate. Lipase I, the first eluted fraction, was the predominant form, and more than 80% of the total activity was attributed to this form. Amino acid sequence analysis of the amino and carboxyl termini of these two enzyme preparations indicated that lipase I was the product of the lipase gene whose cDNA had previously been cloned and sequenced [Shimada et al. (1989) J. Biochem. 106, 383-388]. Lipase II, on the other hand, had similar amino acid composition, but different terminal sequences which were not found in the primary structure of lipase I deduced from the cDNA sequence. These results gave lines of evidence for the expression of truely different lipase genes and ruled out the possibility that the observed multiple forms are caused by proteolytic digestion. The molecular mass estimated by SDS-PAGE and the isoelectric point of lipase I were 64 kDa and 4.3, while those of lipase II were 66 kDa and 4.3, respectively. The two lipases had essentially the same specific activities, substrate specificities, pH stabilities, and optimal temperatures, but different pH optima and thermal stabilities.     

Lipase in lipid bodies of cotyledons of rape and mustard seedlings

Lipolytic activity was absent in the crude cotyledon extract of ungerminated rapeseed (Brassica napm L. var. Dwarf Essex), and increased to a peak at day 4 in seedling growth, concomitant with the decrease in total lipids. About 50% of the lipase activity was recovered in the lipid bodies isolated from the cotyledon extract by flotation centrifugation. Isolated lipid bodies underwent autolysis of internal triacylglycerols resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies had been extracted with diethyl ether, the lipase was recovered in the remaining membrane fraction. The lipase had a maximal activity at pH 6.5 on trierucin, trilinolein, or endogenous triacylglycerols, and at pH 8.0 on N-methylindoxylmyristate. The lipase was most active on trierucin and trilinolein, and hydrolyzed the related di- and monoacylglycerols at lower rates. There was little enhancement of the lipase activity in the presence of NaCl, CaCl₂, or detergents, and detergents in general reduced the activity. The hydrolysis of trierucin was linear until about 50% of the trierucin had been converted to erucic acid, and there was little accumulation of dierucin and monoerucin. Lipase extracted from lipid bodies isolated from germinated rapeseed of the variety Tower, which contains little or no erucic acids in the storage triacylglycerols, also had the highest activities on trierucin and trilinolein. A comparative study on mustard seed (Brassica juncea) revealed that the mustard lipase possessed characteristics very similar to those of the rapeseed lipase.     

Biosynthesis of lipase in the scutellum of maize kernel

In the scutellum of maize kernel after imbibition, lipase activity increased rapidly, concomitant with the decrease in storage triacylglycerols. The enzyme activity peaked at day 6, but remained at the same level from day 6-10 when most of the triacylglycerols had been depleted. By in vitro translation with extracted RNAs followed by immunoprecipitation, and by resolving the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lipase was found to be de novo synthesized in postgermination. The enzyme was synthesized by RNAs extracted from free polyribosomes and not from bound polyribosomes. Both in vitro and in vivo synthesized lipase had the same Mr of 65,000 as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as had the purified authentic enzyme; thus there was no appreciable co- or post-translational processing of the enzyme. Lipase-specific mRNA was present only between day 2-6 after imbibition. At day 6 when lipolysis was most active, more than 60% of the lipase activity was recovered in the lipid body fraction and specifically associated with the organelle membrane. From day 6-10, the lipase activity gradually shifted from the lipid body fraction to other subcellular fractions, including the 10,000 X g pellet, the 120,000 X g pellet, and the 120,000 X g supernatant. Lipase in these subcellular fractions was attributed to represent the enzyme associated with membrane ghosts of the lipid bodies which were fusing with the fragile cell vacuoles; such fusions were observed in situ by electron microscopy.     

Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings

Until now, there has been no conclusive demonstration of any in vivo oleosin degradation at the early stages of oil body mobilization. The present work on sunflower (Helianthus annuus L.) has demonstrated limited oleosin degradation during seed germination. Seedling cotyledon homogenization in Tris-urea buffer, followed by SDS-PAGE, revealed three oleosins (16, 17.5 and 20 kDa). Incubation of oil bodies with total soluble protein from 4-day-old seedlings resulted in oleosin degradation. In vitro and in vivo degradation of the 17.5-kDa oleosin was faster than the other two, indicating its greater susceptibility to proteolysis. Oleosin degradation by the total soluble protein resulted in a transient 14.5-kDa polypeptide, followed by an 11-kDa protease-protected fragment, which appeared post-germinatively and accumulated corresponding to increased rate of lipid mobilization. A 65-kDa protease, active at pH 7.5-9.5, was zymographically detected in the total soluble protein. Its activity increased along with in vivo accumulation of the protease-protected fragment during seed germination and accompanying lipid mobilization. Protease-treated oil bodies were more susceptible to maize lipase action. Differential proteolytic sensitivity of different oleosins in the oil body membranes could be a determinant of oil body longevity during seed germination.     

奶牛瘤胃微生物元基因组文库中脂肪酶的筛选与酶学性质

利用含有三油酸甘油酯的脂肪酶选择性筛选培养基,从奶牛瘤胃微生物元基因组文库15360个克隆中,筛选得到了18个脂肪酶阳性克隆,其插入片段大约为60kb,并且各个克隆的插入片段各不一样。利用p-NPP法对脂肪酶克隆的脂肪酶活性分析,表明均具有大小不等的脂肪酶活性。底物特异性分析表明Lipase6、Lipase7和Lipase8分别对C16底物(对硝基苯棕榈酸酯)、C12底物(对硝基苯月桂酸酯)和C16底物(对硝基苯棕榈酸酯)水解能力最强。Lipase6、Lipase7、Lipase8的脂肪酶最适pH为7.5;Lipase8的脂肪酶活性半衰期随反应温度的升高而缩短,70oC时能达到30min。本研究所筛选的脂肪酶具有不同的底物特异性和较好的热稳定性,这对于工业化生产具有一定的应用潜力。     

Lipase Activities in Castor Bean Endosperm during Germination

Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was located mainly in glyoxysomes, with some 30% of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal “ghosts” at equilibrium density 1.21 g/cm³ on the sucrose gradient. Association of the lipase with the gly-oxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.     

Esterase and lipase activity in Jatropha curcas L. seeds

Two new esterases (JEA and JEB) and a lipase (JL) were extracted from the seeds of Jatropha curas L. Lipase activity was only found during germination of the seeds and increased to a maximum after 4 days of germination. All enzymes were found to be most active in the alkaline range at around pH 8 and the purified (fractionated precipitation with ethanol and gel filtration) esterases were very stable at high temperatures. The molecular weight (SDS-PAGE) of both esterases was determined to be 21.6-23.5 kDa (JEA) and 30.2 kDa (JEB) and the isoelectric point was 5.7-6.1 for esterase JEA and 9.0 for esterase JEB. Most ions caused a negative influence on the activity of both esterases. Using p-nitrophenyl butyrate as a substrate JEA showed a K(m) of 0.02 mM and a v(max) of 0.26 micromol mg(-1) min(-1). Under the same conditions JEB showed a K(m) of 0.07 mM and a v(max) of 0.24 micromol mg(-1) min(-1). Both esterases hydrolyzed tributyrin, nitrophenyl esters up to a chain length of =C4 and naphtylesters up to a chain length =C6. In transesterification reactions, JL was found to be most active at very low water activities (0.2) and in high water activities, the lipase hydrolysed triglycerides into conversions above 80%. The lipase hydrolysed both short chain and long chain triglycerides at about the same rate but was inactive on alpha-methylbenzyl acetate. JL is a potentially useful biocatalyst in the hydrolysis of triglycerides in organic solvents.     

Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1

A thermophilic microorganism, Bacillus thermoleovorans ID-1, isolated from hot springs in Indonesia, showed extracellular lipase activity and high growth rates on lipid substrates at elevated temperatures. On olive oil (1.5%, w/v) as the sole carbon source, the isolate ID-1 grew very rapidly at 65 degrees C with its specific growth rate (2.50 h(-1)) and its lipase activity reached the maximum value of 520 U l(-1) during the late exponential phase and then decreased. In addition to this, isolate ID-1 could grow on a variety of lipid substrates such as oils (olive oil, soybean oil and mineral oil), triglycerides (triolein, tributyrin) and emulsifiers (Tween 20, 40). The excreted lipase of ID-1 was purified 223-fold to homogeneity by ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography and Sephacryl S-200 gel filtration chromatography. As a result, the relative molecular mass of the lipase was determined to be 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed optimal activity at 70-75 degrees C and pH 7.5 and exhibited 50% of its original activity after 1 h incubation at 60 degrees C and 30 min at 70 degrees C and its catalytic function was activated in the presence of Ca(2+) or Zn(2+).     

Acid Lipase of Castor Bean Lipid Bodies : Isolation and Characterisation

The acid lipase of castor endosperm lipid bodies has been studied using colorimetric assay based on the measure of the hydrolytic activity of p-nitrophenyl ester of palmitate and other acyl derivatives. These substrates are compatible with the natural triacylglycerols for the measure of lipolytic activities. The subcellularly-surveyed acid lipolytic activity in the germinated castor bean endospermal tissue was found to be enhanced in the lipid bodies. The lipase, which is partially latent and tightly associated with lipid bodies, is an exceptionally stable enzyme with an optimum activity at pH 4.5 and displays an inverse relationship between its activity and the acyl chain length of its substrate. To facilitate isolation of the acid lipase, a procedure has been developed to solubilise the membrane-bound enzyme in an active form. The detergent-solubilised acid lipase after two chromatographic steps yielded an eight-fold active preparation which after gel permeation resolved as heterogeneous aggregate in excess of 500 kD. Lipase-enriched preparations showed consistent presence of 14 and 60 kD proteins which constituted the most abundant species of the lipid bodies. Although it has not been possible to obtain an active lipase preparation in a state free of either the 14 or 60 kD protein, the lipase activity in the detergent extracts of lipid bodies was immunoprecipitable with antibodies raised against the 60 kD component.     

Lipase H,a new member of the triglyceride lipase family synthesized by the intestine

We report here the molecular cloning of a novel member of the triglyceride lipase family, a 2.4-kb cDNA encoding human lipase H (LIPH) and the mouse ortholog (Liph). The human LIPH cDNA encodes a 451-amino-acid protein with a lipase domain. Mouse Liph shows 85% amino acid identity and 75% nucleotide identity to human LIPH. Human LIPH exhibits 47% identity with phosphatidylserine-specific phospholipase A1 (PS-PLA1) and 46% identity with endothelial lipase (LIPG) and lipoprotein lipase (LPL). LIPH is localized on human chromosome 3q27-q28. Northern blot analysis revealed specific expression of LIPH mRNA in intestine, lung, and pancreas. Lipase H protein was also detected in human intestine. Lipase H is a secreted protein with an apparent molecular weight of 63 kDa. Although several lipid substrates were tested, the lipid substrate of LIPG was not identified. Like the other members of this gene family, LIPH may be involved in lipid and energy metabolism.     

Purification and characterization of a lipase from the glycolipid-producing yeast Kurtzmanomyces sp. I-11

An extracellular lipase produced by the glycolipid-producing yeast Kurtzmanomyces sp. I-11 was purified by ammonium sulfate precipitation and column chromatographies on DEAE-Sephadex A-25, SP-Sephadex C-50, and Sephadex G-100. Based on the analysis of the purified lipase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified lipase was judged to be homogeneous and its molecular mass was estimated to be approximately 49 kDa. The optimum temperature for the activity was 75 degrees C, and the activity was very stable at temperatures below 70 degrees C. The active pH range of this lipase was 1.9-7.2, and the activity was stable at pH below 7.1. The lipase showed a preference for C18 acyl groups by measurements with p-nitrophenyl esters and triglycerides as substrates. The lipase was very stable in the presence of various organic solvents at a concentration of 40%. Although the N-terminal sequence of the Kurtzmanomyces lipase was very similar to that of lipase A from Candida antarctica, the pH profiles of the two lipases were significantly different.     

Purification and characterization of two distinct lipases from Geotrichum candidum

Lipase, an enzyme that hydrolyzes triacylglycerol, has been purified and characterized. The purification procedure includes ethanol precipitation and chromatographies on Sephacryl-200 HR, high resolution anion-exchange (mono Q) and Polybuffer exchanger 94. With this procedure, two forms of lipases from Geotrichum candidum were obtained. Lipase I (main enzyme) and lipase II (minor enzyme) were purified 35-fold with a 62% recovery in activity and 94-fold with a 18% recovery in activity, respectively. Their molecular weights have been estimated by polyacrylamide gel electrophoresis under denaturing conditions and by molecular sieving under native conditions at 56,000. Lipase I and II had optimum pH values of 6.0 and 6.8 and isoelectric points of 4.56 and 4.46, respectively. The enzymes are stable at a pH range of 6.0 to 8.0. Monovalent ions had little effect on both enzyme activities, while divalent ions at concentrations above 50 mM inhibited the lipase activities in a concentration-dependent manner. Sodium dodecyl sulfate at a concentration lower than 10 mM completely inhibited the lipase activity.     

Ultrastructure and Lipase Activity of Cotyledon cell During Pod Development in Peanut

A series of significant changes of the ultrastructure and lipase activity of cotyledon cell were found in peanut (Arachis hypogaea) during pod development. In he initial stage of cotyledon development there were many plastids which kept producing starch grain and there were low lipase activity and very few lipid and protein bodies in the cell. In the middle stage of cotyledon development, a great number of larger lipid bodies were seen in the cell and a lot of protein bodies formed in the vacuoles and continued to increase in size. Lipase activity increased in the cytoplasm, endoplasmic reticulum, protein bodies, plasmalemma and intercellular space. In the later stage of cotyledon development, the lipid bodies did not increase in number but became slightly larger. The protein bodies continued to increase both in number and in size. Lipase acttvity was even hegher in the cytoplasm. In the final stage the protein bodies became irregular in shape and some of them tended to disintegrate with their content entered into the space around the lipid bodies. The lipase activity in the cell declined. The results indicated that the lipid body originated in the cytoplasm and the protein body originated in the vacuole; that the accumulation of oil and protein in peanut cotyledon resulted from the formation and development of lipid and protein bodies in the cell, and that the changes of plasmid and lipase activity in the cell played a role in the development of lipid body during the development of cotyledon.     

花生荚果发育过程中子叶细胞的超微结构和脂酶活性的研究

在花生(Arachis hypogaea)荚果发育过程中,子叶细胞的超微结构和脂酶活性皆发生了显著变化。子叶生长初期,缅胞中质体较多,并不断形成淀粉粒;脂酶活性低,脂体和蛋白体很少。子叶发育中期,子叶细胞质中出现大量体积较大的脂体,液泡中的蛋白体不断形成和增大,而且细胞质、内质网、蛋白体外膜、细胞质膜和细胞间隙上皆显示较强的脂酶活性。子叶发育后期,脂体数量不再增加,但体积略有增大,间质透明度也有提高;蛋白体增大较小,但数量却进一步增多;细胞质中仍显示较强的脂酶活性。至末期时,蛋白体形态变得不规则,甚至出现部分解体,其基质充挤脂体间隙;细咆中的脂酶活性减弱。研究表明,花生脂体起源于细胞质,蛋白体起源于液泡,子叶油分和蛋白质的积累足体内脂体和蛋白体不断发育的结果,细胞中脂酶活性的变化可能与脂体发育有关。     

Purification and general properties of a metal-insensitive lipase from Rhizopus japonicus NR 400

Lipase [triacylglycerol lipase, EC 3.1.1.3] has been purified to homogeneity from Rhizopus japonicus NR 400 by chromatography on hydroxylapatite, octyl-Sepharose and Sephacryl S-200. It showed a molecular weight of about 30,000 by SDS-PAGE and a specific activity of 68,900 units/mg protein. The enzyme catalyzed the hydrolysis of tricapryn and tricaprylin rapidly in comparison with other triglycerides. This lipase had an optimum pH of around 5, and albumin enhanced its activity between pH 3 and 8. The composition of fatty acids liberated from linseed oil by the lipase was similar to that in the case of pancreatic lipase. The lipase activity was not affected by the addition of 1 mM metal ions or bile salts. Stimulation of the lipase activity was observed upon addition of albumin to the reaction mixture. Immunotitration experiments were also performed with antibodies raised against the purified lipase.     

Electric field control of enzyme membrane activity.

Lipase was immobilized in liquid crystal-collagen membrane. No difference in the optimum pH between the lipase-liquid crystal-collagen membrane and native lipase was observed. However, the immobilized lipase shows flat-pH profiles. The activity of the lipase membrane on a platinum electrode used for cathode increased with increase in terminal voltage and then returned to the initial activity with decrease of terminal voltage.     

Regulation of lipases by lipid-lipid interactions: implications for lipid-mediated signaling in cells

Lipases are extracellular peripheral proteins that act at the surface of lipid emulsions stabilized, typically, by phospholipids. At a critical composition lipase activity toward substrates in phospholipid monolayers is discontinuously switched on by a small increase in substrate mole fraction. This occurs in part because lipase binding is inhibited by phospholipids. Binding of the lipase cofactor, colipase, is also inhibited by phospholipids. The initial rate of colipase binding increases abruptly at a substrate mole fraction that is approximately half the critical composition for lipase activity and just above that in substrate-phospholipid complexes. Moreover, complex collapse areas show an approximately 1:1 correlation with phospholipid excluded areas determined from an analysis of colipase adsorption rates. Thus, complexes inhibit colipase binding rate. Additionally, the switching of lipase activity likely occurs when uncomplexed substrate becomes the majority species in the interface. Lipase substrates, e.g. diacylglycerols, are typically the same lipids generated in the cytoplasmic surface of the plasma membrane of stimulated cells. As colipase binding is nonspecific and complexes involving lipase substrates form on the basis of lipid-lipid interactions alone, complexes should form in the plasma membrane of stimulated cells and may regulate protein translocation to the membrane.