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1.
Synthesis and secretion of active lipoprotein lipase in Chinese-hamster ovary (CHO) cells. 总被引:1,自引:0,他引:1
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Cultured Chinese-hamster ovary cells (CHO cells) were found to produce and secrete a lipase, which was identified as a lipoprotein lipase by the following criteria. Its activity was stimulated by serum and apolipoprotein CII, and was inhibited by high salt concentration. The lipase bound to heparin-agarose and co-eluted with 125I-labelled bovine lipoprotein lipase in a salt gradient. A chicken antiserum to bovine lipoprotein lipase inhibited the activity and precipitated a labelled protein of the same apparent size as bovine lipoprotein lipase from media of CHO cells labelled with [35S]methionine. The lipase activity and secretion were similar in growing cells and in cells that had reached confluency. Hence, lipoprotein lipase appears to be expressed constitutively in CHO cells and is not linked to certain growth conditions, as in pre-adipocyte and macrophage cell lines. At 37 degrees C, but not at 4 degrees C, heparin increased the release of lipase to the medium 2-4-fold. This increased release occurred without depletion of cell-associated lipase activity, suggesting that heparin enhanced release of newly synthesized lipase. 相似文献
2.
Summary Production of lipase by free and alginate-entrapped protoplasts was studied in batch culture. Cell-wall-degrading enzymes Novozym 234 and cellulase CP improved lipase secretion of normal mycelium by 25%–100%. The protoplast-regenerated mycelium exhibited several-fold higher lipase activity in batch replacements in TRIS buffer over normal spore-derived mycelium. The specific lipase activity of immobilized protoplasts was about four times higher than normal mycelial beads. Protoplasts beads were stable and retained high enzyme activity even after three buffer replacements lasting 120 h; TRIS buffer was better than acetate or normal glucose medium. A minimum of 8 h regeneration period was necessary for lipase synthesis. Triolein, olive oil, tributyrin and oleic acid butylester were able to induce lipase in immobilized protoplasts. Tween 80 enhanced lipase activity of the immobilized protoplasts. Partially degraded immobilized mycelium was nearly as effective as normal immobilized protoplasts for lipase secretion. Both free and immobilized protoplasts could be reused for up to 200 h with some loss in enzyme activity. 相似文献
3.
Four racemic esters of (1R*,7aR*)-3,6,7,7a-tetrahydro-1-hydroxy-7a-methyl-1H-inden-5(2H)-one were prepared and subjected to hydrolysis with two types of hydrolases, including alcalase and three lipases. Alcalase and lipase showed opposite enantiopreference on these esters. Based on this result, we developed a gram-scale procedure using butanoate as the substrate, which was treated consecutively with alcalase and lipase from Candida rugosa (CRL), to give both enantiomers of the title compound in high yields and high enantiomeric excess. 相似文献
4.
A facile chemoenzymatic synthesis of both the S and R forms of 5-(1-aminoethyl)-2-(cyclohexylmethoxy)benzamide a key intermediate of non-peptidic Src SH2 inhibitors is described. Both the enantiomers were synthesized in high optical purity (>99% ee) by reduction followed by lipase-mediated acylation of the precursor 6 in one-pot. Immobilized Pseudomonas cepacia lipase offered high degree of enantioselectivity with spontaneity. 相似文献
5.
The cultivation conditions of wild-type strain V-10 and mutant strain M-1 (overproducer of endonuclease and chitinase) of Serratia marcescens optimal for extracellular lipase biosynthesis were determined. The strain V-10 displayed the maximal lipase yield (840 AU/ml) after 10-12 h of cultivation; the strain M-1 (33 AU/ml), after 25-30 h. The data showed that extracellular lipases from V-10 and M-1 can be precipitated in a weakly acid medium (pH 5.0 and 4.5, respectively). This property was used to obtain partially purified lipase preparations. The effect of the ionic composition of the reaction mixture on the activities of these enzymatic preparations was studied. Both preparations displayed highest activities in weakly alkaline media (pH 8.0); however, the wild-type strain lipase displayed a higher thermal stability and stability at alkaline pH compared with M-1 lipase. Both lipases were activated by various anionic and nonionic surfactants and inactive in the presence of cetyltrimethylammonium bromide. 相似文献
6.
An aliphatic polythioester was enzymatically prepared by the direct polycondensation of mercaptoalkanoic acid using immobilized lipase of Candida antarctica (lipase CA) in bulk. The commercially available 11-mercaptoundecanoic acid was polymerized by lipase CA in bulk in the presence of molecular sieves 4A as a water absorbent at 110 degrees C for 48 h to produce poly(11-mercaptoundecanoate) with an M(w) of 34 000 in high yield. The 104.5 degrees C melting temperature (T(m)) of poly(11-mercaptoundecanoate) was about 20 degrees C higher than that of the corresponding polyoxyester, poly(11-hydroxyundecanoate). The polythioester was readily transformed by lipase into the corresponding cyclic oligomers mainly consisting of the dimer, which were readily repolymerized by the ring-opening polymerization using lipase as a sustainable chemical recycling. 相似文献
7.
A. B. Duzhak Z. I. Panfilova E. A. Vasyunina 《Applied Biochemistry and Microbiology》2000,36(4):344-352
—The cultivation conditions of wild-type strain V-10 and mutant strain M-l (overproducer of endonuclease and chitinase) ofSerratia marcescens optimal for extracellular lipase biosynthesis were determined. The strain V-10 displayed the maximum lipase yield (840 AU/ml)
after 10–12 h of cultivation; the strain M-l (330 AU/ml), after 25–30 h. The data showed that extracellular lipases from V-10
and M-1 can be precipitated in a weakly acidic medium (pH 5.0 and 4.5, respectively). This property was used to obtain partially
purified lipase preparations. The effect of the ionic composition of the reaction mixture on the activities of these enzymatic
preparations was studied. Both preparations displayed the highest activities in weakly alkaline media (pH 8.0); however, the
wild-type strain lipase displayed higher thermal stability and stability at alkaline pH compared with M-1 lipase. Both lipases
were activated by various anionic and nonionic surfactants and were inactive in the presence of cetyltrimethylammonium bromide. 相似文献
8.
The enzymatic degradation and repolymerization were carried out with the objectives of developing the chemical recycling of aliphatic polyester-type plastics, such as the poly(butylene adipate) (PBA), poly(butylene succinate), and poly(butylene adipate-co-succinate) copolymers which are typical biodegradable plastics. They were degraded by lipase in an organic solvent solution containing a small amount of water to produce cyclic oligomers mainly consisting of the cyclic diester. The produced cyclic oligomer was readily repolymerized by lipase to produce a polyester having an equal or higher molecular weight compared to the parent polymer. As an example, PBA having an Mw of 22,000 was almost quantitatively transformed by lipase CA (Novozym 435) in water-containing toluene at 50 degrees C into the corresponding cyclic oligomers mainly consisting of dimers. Thus, the obtained oligomers were readily polymerized by lipase CA to produce the PBA with an Mw of 52,000. 相似文献
9.
Hugo J. Gorissen Jean-Pierre Van Hoeck Anne M. Mockel Guido H. Journe Claude Delatour Valry R. Libert 《Chirality》1992,4(5):286-294
Both (R)- and (S)-4-hydroxypentylaminoacetamide have been synthesized by reductive amination of glycinamide on the γ-valerolactols corresponding to (R)- and (S)-γ-valerolactone, respectively. These enantiomeric lactones were readily obtained in high enantiomeric excess (ee) by enzymic porcine pancreatic lipase (PPL) kinetic resolution of rac-methyl γ-hydroxyvalerate. © 1992 Wiley-Liss, Inc. 相似文献
10.
Perilipin Controls Lipolysis by Regulating the Interactions of AB-hydrolase Containing 5 (Abhd5) and Adipose Triglyceride Lipase (Atgl) 总被引:1,自引:0,他引:1
James G. Granneman Hsiao-Ping H. Moore Rukmani Krishnamoorthy Miloni Rathod 《The Journal of biological chemistry》2009,284(50):34538-34544
The mobilization of stored lipid by hormones is a fundamental function of fat cells, and there is strong evidence that perilipin (Plin), a lipid droplet scaffold, and adipose tissue triglyceride lipase (Atgl), a triglyceride-specific lipase, play critical roles. Previous work suggested that Abhd5, a protein activator of Atgl, coordinates with Plin in controlling basal and stimulated lipolysis; however, the underlying mechanism is controversial. The present experiments investigated protein trafficking and interactions among Plin, Atgl, and Abhd5 in live cells. The results demonstrate that Plin binds Abhd5 with high affinity and thereby suppresses the interaction of Abhd5 with Atgl. Sequestration of Abhd5 appears to a major mechanism by which Plin reduces basal lipolysis. Phosphorylation of Plin on serine 492 or serine 517 rapidly releases Abhd5 from Plin, allowing Abhd5 to directly interact with Atgl. Imaging experiments demonstrated that the Plin-dependent interaction of Abhd5 and Atgl occurs mainly, but not exclusively, on lipid droplets that contain Plin. 相似文献
11.
A straightforward approach to enantiomerically enriched (R) and (S)-3-hydroxycyclopentanone is described. The key step involves a kinetic resolution of racemic 3-hydroxycyclopentanone using commercial Pseudomonas cepacia lipase immobilized on diatomite (Amano lipase PS-DI). The absolute stereochemistry of the product was determined by derivatization into (R)-3-(benzyloxy)cyclopentanone. 相似文献
12.
13.
脂肪组织甘油三酯水解酶(adipose triglyceride lipase, ATGL)是一种催化甘油三酯第一步水解的重要脂肪酶,在机体能量代谢调节中发挥重要作用.本文介绍了ATGL的基因和蛋白质结构,并详细综述了ATGL的功能调控和与其相关联疾病的研究进展,最后通过与激素敏感脂肪酶(HSL)比较,对ATGL的特征进行总结. 相似文献
14.
Rauda A. Mohamed Abu Bakar Salleh Adam Thean Chor Leow Normi M. Yahaya Mohd Basyaruddin Abdul Rahman 《Molecular biotechnology》2017,59(7):284-293
An enzyme with broad substrate specificity would be an asset for industrial application. T1 lipase apparently has the same active site residues as polyhydroxyalkanoates (PHA) depolymerase. Sequences of both enzymes were studied and compared, and a conserved lipase box pentapeptide region around the nucleophilic serine was detected. The alignment of 3-D structures for both enzymes showed their active site residues were well aligned with an RMSD value of 1.981 Å despite their sequence similarity of only 53.8%. Docking of T1 lipase with P(3HB) gave forth high binding energy of 5.4 kcal/mol, with the distance of 4.05 Å between serine hydroxyl (OH) group of TI lipase to the carbonyl carbon of the substrate, similar to the native PhaZ7 Pl . This suggests the possible ability of T1 lipase to bind P(3HB) in its active site. The ability of T1 lipase in degrading amorphous P(3HB) was investigated on 0.2% (w/v) P(3HB) plate. Halo zone was observed around the colony containing the enzyme which confirms that T1 lipase is indeed able to degrade amorphous P(3HB). Results obtained in this study highlight the fact that T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation activity but amorphous P(3HB) degradation activity as well. 相似文献
15.
Goksel N. Demirer Metin Duran Robert D. Tanner 《Biotechnology and Bioprocess Engineering》1996,1(1):46-50
In order to design industrial scale reactors and proceises for multi-phase biocatalytic reactions, it is essential to understand the mechanisms by which such systems operate. To illustrate how such mechanisms can be modeled, the hydrolysis of the primary ester groups of triglycerides to produce fatty acids and monoglycerides by lipase (glycerol-ester hydrolase) catalysis has been selected as an example of multiphase biocatalysis. Lipase is specific in its behavior such that it can act only on the hydrolyzed (or emulsified) part of the substrate. This follows because the active center of the enzyme is catalytically active only when the substrate contacts it in its hydrolyzed form. In other words, lipase acts only when it can shuttle back and forth between the emulsion phase and the water phase, presumably within an interphase or boundary layer between these two phases. In industrial applications lipase is employed as a fat splitting enzyme to remove fat stains from fabrics, in making cheese, to flavor milk products, and to degrade fats in waste products. Effective use of lipase in these processes requires a fundamental understanding of its kinetic behavior and interactions with substrates under various environmental conditions. Therefore, this study focuses on modeling and simulating the enzymatic activity of the lipase as a step towards the basic understanding of multi-phase biocatalysis processes. 相似文献
16.
Igor Hernndez Mertxe de Renobales Mailo Virto Francisco J. Prez-Elortondo Luis J.R. Barron Cristian Flanagan Marta Albisu 《Enzyme and microbial technology》2005,36(7):870-879
Three commercial lipases (lipases A1 and A2 were pregastric lipases and lipase A3 was a fungal lipase) from three different manufacturers were studied to develop the characteristic pungent flavour of Idiazabal (sheep's raw milk) cheese in experimental productions (50 L vats), both in summer and in winter. In the experimental productions, all lipases significantly increased the concentration of total free fatty acids (FFA), both after 90 and 180 days of ripening. Lipases A1 and A2 increased primarily the concentration of short-chain FFA (C4, C6 and C10), whereas C16:0 and C18:1 were the main FFA obtained with lipase A3. Cheeses made with no lipase had the lowest concentrations of total FFA, with C16:0 and C18:1 as the major FFA. The percent of short-chain FFA was 70–80% of the total for lipases A1 and A2, but 30–40% in cheeses made with lipase A3 or with no lipase. Sensory analysis was performed after 90 and 180 days of ripening. Cheeses made with lipases A1 or A2 or with lamb rennet paste (which contains pregastric lipase and was used as control for sensory analysis) had significantly higher scores in 6 of the 22 attributes analyzed (odour and flavour intensity, sharp odour, pungent flavour and rennet odour and flavour) than cheeses made with lipase A3 or with no lipase added. Lipase A1 was selected to conduct commercial cheese productions (200 L vats) made by artisan cheese makers adding 94 lipase units (LU)/50 L (high amount) or 8 LU/50 L (low amount). Control cheeses were made with lamb rennet paste having comparable amounts of lipolytic activity. After 90 and 180 days of ripening, cheeses made with the same amount of lipase (either as lipase A1 or present in lamb rennet paste) were indistinguishable from each other, both from the sensory and analytical points of view (comparable amounts of total FFA, and percent FFA composition). Those with low amount of lipase were rated as ‘mild’, whereas those with high amount of lipase were rated as ‘strong’. An industrial production (5000 L vat) with 100 LU/50 L yielded cheeses of ‘intermediate strength’ and intermediate levels of lipolysis. A linear correlation was observed between percent of short-chain FFA and the score for pungent flavour, for all amounts of lipase used in this study. We conclude that lipase A1 is an adequate commercial lipase to develop the characteristic flavour of Idiazabal cheese, both in artisan and industrial fabrications. 相似文献
17.
Lipase-catalyzed enantioselective esterification of (S)-naproxen hydroxyalkyl ester in organic media
A lipase-catalyzed, enantioselective esterification process in organic solvents was developed for the synthesis of (S)-naproxen hydroxyalkyl ester. With the selection of lipase (Candida rugosa lipase) and reaction medium (isooctane and cyclohexane), a high enantiomeric ratio of <100 for the enzyme was obtained. 1,4-Butanediol was the best acyl acceptor. The carbon chain length of the alcohol had a major effect on the enzyme activity and enantioselectivity of lipase-catalyzed esterification. 相似文献
18.
Olle Hernell Torbjörn Egelrud Thomas Olivecrona 《Biochimica et Biophysica Acta (BBA)/General Subjects》1975,381(2):233-241
A rabbit antiserum prepared against the serum-stimulated lipase (lipoprotein lipase) from bovine milk crossreacted with serum-stimulated lipases from human milk and from human postheparin plasma, but not with bile salt-stimulated lipase from human milk or with salt-resistant lipase from human postheparin plasma. Thus, the serum-stimulated lipase in bovine milk has immunological determinants in common with the serum-stimulated lipases in human milk and in human postheparin plasma. The time-courses for the appearance of serum-stimulated lipase and salt-resistant lipase activities in human plasma after heparin injection were different. The two activities were separated by heparin-Sepharose chromatography. After treatment of postherapin plasma with the antiserum only the salt-resistant lipase activity could be eluted from the column. Thus, these two enyzme activities in postheparin plasma reside in two different enzyme molecules. 相似文献
19.
cDNAs encoding rat adipose tissue hormone-sensitive lipase were expressed in COS cells, under the control of the SV40 promoter to half the level in rat adipocytes, the richest native source of the enzyme. A cDNA lacking most of the long 5'-untranslated region of the full-length rat hormone-sensitive lipase cDNA was, with regard to the lipase activity, on the average 70% more efficiently expressed that the full-length cDNA. The recombinant protein was almost identical to hormone-sensitive lipase of rat adipose tissue with respect to specific activity, susceptibility to inhibitors, molecular size, phosphorylation and activation by cyclic AMP-dependent protein kinase. The described eukaryotic expression system will allow analysis of effects of amino acid substitutions introduced into the lipase molecule by site-directed mutagenesis. 相似文献