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51.
52.
Neuropathy target esterase (NTE) is an integral membrane protein localized in the endoplasmic reticulum in neurons. Irreversible inhibition of NTE by certain organophosphorus compounds produces a paralysis known as organophosphorus compound-induced delayed neuropathy. In vitro, NTE has phospholipase/lysophospholipase activity that hydrolyses exogenously added single-chain lysophospholipids in preference to dual-chain phospholipids, and NTE mutations have been associated with motor neuron disease. NTE's physiological role is not well understood, although recent studies suggest that it may control the cytotoxic accumulation of lysophospholipids in membranes. We used the NTE catalytic domain (NEST) to hydrolyze palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (p-lysoPC) to palmitic acid in bilayer membranes comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the fluorophore 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC). Translational diffusion coefficients (DL) in supported bilayer membranes were measured by fluorescence recovery after pattern photobleaching (FRAPP). The average DL for DOPC/p-lysoPC membranes without NEST was 2.44 µm2s-1 ± 0.09; the DL for DOPC/p-lysoPC membranes containing NEST and diisopropylphosphorofluoridate, an inhibitor, was nearly identical at 2.45 ± 0.08. By contrast, the DL for membranes comprising NEST, DOPC, and p-lysoPC was 2.28 ± 0.07, significantly different from the system with inhibited NEST, due to NEST hydrolysis. Likewise, a system without NEST containing the amount of palmitic acid that would have been produced by NEST hydrolysis of p-lysoPC was identical at 2.26 ± 0.06. These results indicate that NTE's catalytic activity can alter membrane fluidity. 相似文献
53.
Teresa Matamá Rita Araújo Georg M. Gübitz Margarida Casal Artur Cavaco‐Paulo 《Biotechnology progress》2010,26(3):636-643
In the present work, we describe for the first time the specific role of cutinase on surface modification of cellulose acetate fibers. Cutinase exhibits acetyl esterase activity on diacetate and triacetate of 0.010 U and 0.007 U, respectively. An increase on the hydroxyl groups at the fiber surface of 25% for diacetate and 317% for triacetate, after a 24 h treatment, is estimated by an indirect assay. Aiming at further improvement of cutinase affinity toward cellulose acetate, chimeric cutinases are genetically engineered by fusing the 3′‐end coding sequence with a bacterial or a fungal carbohydrate‐binding module and varying the linker DNA sequence. A comparative analysis of these genetic constructions is presented showing that, the superficial regeneration of cellulose hydrophilicity and reactivity on highly substituted cellulose acetates is achieved by chimeric cutinases. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 相似文献
54.
Pollero Ricardo J. Gaspar María L. Cabello Marta 《World journal of microbiology & biotechnology》2001,17(8):805-810
Several properties of the lipolytic activity exhibited by the conidial fungus Phoma glomerata were studied. Lipolytic activity in an aqueous buffer medium was measured on triacylglycerol, phosphoglyceride and cholesterol ester under different experimental conditions. The effect of storage temperature on the stability of the hydrolytic activity, and optimal conditions of temperature and time of maximal activity were determined. The optimal conditions for maximal lipolytic activity were found to be 40–50 °C and 1 h. The activity released to the medium by 1 mg cells for 1 h at 40 °C was stated as the enzyme released unit (ERU). The protein fraction of MW > 50 kDa obtained by ultrafiltration of the medium, was active on the three substrates assayed, and it showed a non-specific hydrolytic activity on both the 1- and 2-acyl esters either in the neutral glyceride or in the phosphoglyceride. A protein of M
r approx. 75 kDa was the only one that showed esterase activity. The crude medium, stored at –15 °C, maintained its initial hydrolytic activity on triacylglycerol for at least 42 days, though when it was kept for 10 days at 4 °C, the activity fell to 50%. Kinetic parameters using substrates such as triolein (TO), dipalmitoyl phosphatidylcholine (DPPC) and cholesteryl oleate (ChoO), were comparatively evaluated. The activity of the enzyme in the hydrolysis of TO showed the highest values, whereas the maximal specific activities were less when the enzyme was assayed against DPPC and ChoO. 相似文献
55.
Feruloyl esterase (FAE)-catalyzed esterification reaction is as a potential route for the biosynthesis of feruloylated oligosaccharides as functional ingredients. Immobilization of FAE from Humicola insolens on metal chelate-epoxy supports was investigated. The study of effects of immobilization parameters using response surface methodology revealed the significance of enzyme/support ratio (3.25-29.25 mg/g support), immobilization time (14-38 h), buffer molarity (0.27-1.25 M) and pH (4.0-8.0). The interactions between enzyme-to-support ratio/buffer molarity and enzyme-to-support ratio/pH were found to be critical for the modulation of the immobilization activity yield and the retention of specific activity, respectively. Optimum conditions for FAE-immobilization on metal chelate Sepabeads® EC-EP R were identified to be 22.75 mg FAE/g support, pH of 5.0, 27.7 h and buffer molarity of 0.86 M. At these conditions, an activity yield of 82.4%, a specific activity retention of 143.4%, and an enzyme activity of 395.4 μmol/min. g support were achieved. Further incubation of the immobilized FAE at pH 10.0 improved its thermostability. Increasing the pore size of the epoxy support improved the retention of FAE hydrolytic activity and the esterifying efficiency of the immobilized biocatalyst. Optimally immobilized and stabilized FAE on metal chelate-epoxy support retained up to 92.9% of the free enzyme feruloylation efficiency to xylooligosaccharides.. 相似文献
56.
Specific recognition of saturated and 4,5-unsaturated hexuronate sugars by a periplasmic binding protein involved in pectin catabolism 总被引:1,自引:0,他引:1
The process of pectin depolymerization by pectate lyases and glycoside hydrolases produced by pectinolytic organisms, particularly the phytopathogens from the genus Erwinia, is reasonably well understood. Indeed each extracellular and intracellular catabolic stage has been identified using either genetic, bioinformatic or biochemical approaches. Nevertheless, the molecular details of many of these stages remain unknown. In particular, the mechanism and ligand binding profiles for the transport of pectin degradation products between cellular compartments remain entirely uninvestigated. Here we present the structure of TogB, a 45.7 kDa periplasmic binding protein from Yersinia enterocolitica. This protein is a component of the TogMNAB ABC transporter involved in the periplasmic transport of oligogalacturonides. In addition to the unliganded complex (at 2.2 A), we have also determined the structures of TogB in complex with digalacturonic acid (at 2.2 A), trigalacturonic acid (at 1.8 A) and 4,5-unsaturated digalacutronic acid (at 2.3 A). The molecular determinants of oligogalacturonide binding include a novel salt-bridge between the non-reducing sugar uronate group, selectivity for the unsaturated ligand, and the overall sugar configuration. Complementing this are UV difference and isothermal titration calorimetry experiments that highlight the thermodynamic basis of ligand specificity. The ligand binding profiles of the TogMNAB transporter complex nicely complement pectate lyase-mediated pectin degradation, which is a significant component of pectin depolymerization reactions. 相似文献
57.
Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. Here we report the identification of a novel reversible sterol modification in yeast, the sterol acetylation/deacetylation cycle. Sterol acetylation requires the acetyltransferase ATF2, whereas deacetylation requires SAY1, a membrane-anchored deacetylase with a putative active site in the ER lumen. Lack of SAY1 results in the secretion of acetylated sterols into the culture medium, indicating that the substrate specificity of SAY1 determines whether acetylated sterols are secreted from the cells or whether they are deacetylated and retained. Consistent with this proposition, we find that acetylation and export of the steroid hormone precursor pregnenolone depends on its acetylation by ATF2, but is independent of SAY1-mediated deacetylation. Cells lacking Say1 or Atf2 are sensitive against the plant-derived allylbenzene eugenol and both Say1 and Atf2 affect pregnenolone toxicity, indicating that lipid acetylation acts as a detoxification pathway. The fact that homologues of SAY1 are present in the mammalian genome and functionally substitute for SAY1 in yeast indicates that part of this pathway has been evolutionarily conserved. 相似文献
58.
59.
β-Galactosidase (EC: 3.2.1.23), one of the glycosidases detected in Erythrina indica seeds, was purified to 135 fold. Amongst the four major glycosidases detected β-galactosidase was found to be least glycosylated, and was not retained by Con-A CL Seralose affinity matrix. A homogenous preparation of the enzyme was obtained by ion-exchange chromatography, followed by gel filtration. The enzyme was found to be a dimmer with a molecular weight of 74 kDa and 78 kDa, by gel filtration and SDS-PAGE, respectively. The optimum pH and optimum temperature for enzyme activity were 4.4 and 50 °C, respectively. The enzyme showed a Km value of 2.6 mM and Vmax of 3.86 U/mg for p-nitrophenyl-β-D-galactopyranoside as substrate and was inhibited by Zn2+ and Hg2+. The enzyme activity was regulated by feed back inhibition as it was found to be inhibited by β-D-galactose. Chemical modification studies revealed involvement of tryptophan and histidine for enzyme activity. Involvement of tryptophan was also supported by fluorescence studies and one tryptophan was found to be present in the active site of β-galactosidase. Circular dichroism studies revealed 37% α helix, 27% β sheet and 38% random coil in the secondary structure of the purified enzyme. 相似文献
60.
J A Kiernan 《Biotechnic & histochemistry》2007,82(2):73-103
Indoxyl esters and glycosides are useful chromogenic substrates for detecting enzyme activities in histochemistry, biochemistry and bacteriology. The chemical reactions exploited in the laboratory are similar to those that generate indigoid dyes from indoxyl-beta-d-glucoside and isatans (in certain plants), indoxyl sulfate (in urine), and 6-bromo-2-S-methylindoxyl sulfate (in certain molluscs). Pairs of indoxyl molecules released from these precursors react rapidly with oxygen to yield insoluble blue indigo (or purple 6,6'-dibromoindigo) and smaller amounts of other indigoid dyes. Our understanding of indigogenic substrates was developed from studies of the hydrolysis of variously substituted indoxyl acetates for use in enzyme histochemistry. The smallest dye particles, with least diffusion from the sites of hydrolysis, are obtained from 5-bromo-, 5-bromo-6-chloro- and 5-bromo-4-chloroindoxyl acetates, especially the last of these three. Oxidation of the diffusible indoxyls to insoluble indigoid dyes must occur rapidly. This is achieved with atmospheric oxygen and an equimolar mixture of K(3)Fe(CN)(6) and K(4)Fe(CN)(6), which has a catalytic function. H(2)O(2) is a by-product of the oxidation of indoxyl by oxygen. In the absence of a catalyst, the indoxyl diffuses and is oxidized by H(2)O(2) (catalyzed by peroxidase-like proteins) in sites different from those of the esterase activity. The concentration of K(3)Fe(CN)(6)/K(4)Fe(CN)(6) in a histochemical medium should be as low as possible because this mixture inhibits some enzymes and also promotes parallel formation from the indoxyl of soluble yellow oxidation products. The identities and positions of halogen substituents in the indoxyl moiety of a substrate determine the color and the physical properties of the resulting indigoid dye. The principles of indigogenic histochemistry learned from the study of esterases are applicable to methods for localization of other enzymes, because all indoxyl substrates release the same type of chromogenic product. Substrates are commercially available for a wide range of carboxylic esterases, phosphatases, phosphodiesterases, aryl sulfatase and several glycosidases. Indigogenic methods for carboxylic esterases have low substrate specificity and are used in conjunction with specific inhibitors of different enzymes of the group. Indigogenic methods for acid and alkaline phosphatases, phosphodiesterases and aryl sulfatase generally have been unsatisfactory; other histochemical techniques are preferred for these enzymes. Indigogenic methods are widely used, however, for glycosidases. The technique for beta-galactosidase activity, using 5-bromo-4-chloroindoxyl-beta-galactoside (X-gal) is applied to microbial cultures, cell cultures and tissues that contain the reporter gene lac-z derived from E. coli. This bacterial enzyme has a higher pH optimum than the lysosomal beta-galactosidase of animal cells. In plants, the preferred reporter gene is gus, which encodes beta-glucuronidase activity and is also demonstrable by indigogenic histochemistry. Indoxyl substrates also are used to localize enzyme activities in non-indigogenic techniques. In indoxyl-azo methods, the released indoxyl couples with a diazonium salt to form an azo dye. In indoxyl-tetrazolium methods, the oxidizing agent is a tetrazolium salt, which is reduced by the indoxyl to an insoluble coloured formazan. Indoxyl-tetrazolium methods operate only at high pH; the method for alkaline phosphatase is used extensively to detect this enzyme as a label in immunohistochemistry and in Western blots. The insolubility of indigoid dyes in water limits the use of indigogenic substrates in biochemical assays for enzymes, but the intermediate indoxyl and leucoindigo compounds are strongly fluorescent, and this property is exploited in a variety of sensitive assays for hydrolases. The most commonly used substrates for this purpose are glycosides and carboxylic and phosphate esters of N-methylindoxyl. Indigogenic enzyme substrates are among many chromogenic reagents used to facilitate the identification of cultured bacteria. An indoxyl substrate must be transported into the organisms by a permease to detect intracellular enzymes, as in the blue/white test for recognizing E. coli colonies that do or do not express the lac-z gene. Secreted enzymes are detected by substrate-impregnated disks or strips applied to the surfaces of cultures. Such devices often include several reagents, including indigogenic substrates for esterases, glycosidases and DNAse. 相似文献