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1.
The immobilization of pullulanase and beta-amylase on soluble polysaccharides (dextrans and amylose) has been carried out. The method used for coupling the enzymes to the carbohydrate support involves limited periodate oxidation of the polysaccharide followed by reductive alkylation with sodium cyanoborohydride or borohydride. The influence of the degree of functionalization of the carbohydrate, the incubation time, the nature of the reducing agent and, for the dextrans studied, their molecular weight, on the properties of the conjugate were studied. We have observed an apparent correlation between the molecular weight of the glycoprotein conjugates formed and their thermal stability, resistance to urea denaturation and their kinetic parameters. By selecting the proper experimental conditions leading to conjugates with maximum thermal stabilities, it has also been shown that beta-amylase conjugates can hydrolyze starch at a temperature 20 degrees C higher than the corresponding value for the native enzyme. This result demonstrates that conjugation may result in modified enzymes leaving a high operational stability at elevated temperatures. We suggest that the immobilization method presented in this article represents an approach to the stabilization of enzymes employed at an industrial level, which may be of general application. 相似文献
2.
N N Ugarova B M Kershengol'ts I D Artomonov I V Berezin 《Biokhimii?a (Moscow, Russia)》1976,41(10):1829-1836
Pretreatment of peroxidase by its covalent coupling to inert proteins and albumin by means of glutaraldehyde considerably increases the thermostability and specific activity of polyacrylamide gel (PAAG) immobilized peroxidase. The effects of PAAG composition on the catalytic properties of the immobilized oligomers: peroxidase-inert proteins-albumin, are studied. The oligomers immobilized in 40% PAAG (10% N,N'-methylenebisacrylamide) possess the maximal specific activity (4.5 nmol/g). The effects of oligomer composition on their catalytic activity and stability in PAAG are studied. The stability of oligomers of optimal composition (ratio of albumin/peroxidase is 2.4), incorporated into 40% PAAG, is 15 times higher as compared to that of the soluble enzyme and 250 times higher as compared to that of the enzyme incorporated into PAAG without pretreatment. A mechanism of stabilizing effect exerted by albumin on peroxidase in PAAG-immobilized oligomers, is discussed. 相似文献
3.
4.
Öztürk Dilek Coskuner Kazan Dilek Erarslan ltan 《World journal of microbiology & biotechnology》2002,18(9):881-888
The stabilization of Escherichia coli penicillin G acylase (PGA) conjugated with carboxymethylcellulose (CMC) against temperature and pH was studied. The 2,3-dialdehyde derivative of CMC obtained by periodate oxidation was covalently conjugated to PGA via Schiff's base formation. The inactivation mechanism of both native and CMC-conjugated PGA appeared to obey first order inactivation kinetics during prolonged incubations at 40–60 °C and in the pH range 4–9. Inactivation rate constants of conjugated enzyme were always lower, and half-life times were always higher than that of native PGA. The activation free energy of inactivation (G
i values) of CMC-conjugated enzyme were found to be always higher than that of native PGA at all temperatures and pH values studied as another indicator of enzyme stabilization. Highest stability of CMC-conjugated enzyme was observed as nearly four-fold at 40 °C and pH 8.0. No changes were observed on the temperature and pH profiles of PGA after CMC conjugation. Lower K
m and higher k
cat values of PGA obtained after CMC conjugation indicates the improved effect of conjugation on the substrate affinity and catalytic performance of the enzyme. 相似文献
5.
Williams NK Liepinsh E Watt SJ Prosselkov P Matthews JM Attard P Beck JL Dixon NE Otting G 《Journal of molecular biology》2005,346(4):1095-1108
A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10 degrees C by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG=2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding rate of less than 4 min(-1) measured by stopped-flow circular dichroism. The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E.coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding. 相似文献
6.
Enhanced plasma persistence of therapeutic enzymes by coupling to soluble dextran. 总被引:3,自引:0,他引:3
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R F Sherwood J K Baird T Atkinson C N Wiblin D A Rutter D C Ellwood 《The Biochemical journal》1977,164(2):461-464
Conjugation of carboxypeptidase G and arginase, two enzymes of therapeutic interest, to a soluble dextran significantly enhanced plasma persistence in normal and tumour-bearing mice. A prolonged decrease in arginine concentrations in plasma of tumour-bearing mice was demonstrated by using the dextran-linked arginase. Gel filtration of dextran-enzyme conjugate showed that enzyme activity co-chromatographed as a single peak with carbohydrate, and enzyme was shown to be covalently linked to the dextran. 相似文献
7.
Sørensen HP Kristensen JE Sperling-Petersen HU Mortensen KK 《Biochemical and biophysical research communications》2004,319(3):715-719
Ribosomes are extremely soluble ribonucleoprotein complexes. Heterologous target proteins were fused to ribosomal protein L23 (rpL23) and expressed in an rpL23 deficient Escherichia coli strain. This enabled the isolation of 70S ribosomes with covalently bound target protein. Isolation of recombinant proteins from 70S ribosomes was achieved by specific proteolytic cleavage followed by efficient removal of ribosomes by centrifugation. By this procedure we isolated active green fluorescent protein, streptavidin (SA), and murine interleukin-6 (mIL-6). Approximately 500microg of each protein was isolated per gram cellular wet weight. By pull-down assays we demonstrate that SA covalently bound to the ribosome binds d-biotin. Ribosomal coupling is therefore suggested as a method for the investigation of protein interactions. The presented strategy is in particular efficient for the expression, purification, and investigation of proteins forming inclusion bodies in the E. coli cytoplasm. 相似文献
8.
V P Torchilin B A Khaw V N Smirnov E Haber 《Biochemical and biophysical research communications》1979,89(4):1114-1119
A procedure for coupling liposomes to a target specific macromolecular species, anticanine cardiac myosin antibodies, was developed. The anticanine cardiac myosin antibodies covalently coupled to liposomes were shown to retain the specific antibody activity in vitro for the homologous radioiodinated antigen. Radioisotopes can be transported inside such liposome-antibody complexes and were shown experimentally to localize in acute canine myocardial infarction. This new method of coupling of liposome to target-specific agents could provide a novel and highly specific means of transporting radioisotopes or pharmaceuticals protected within the liposomes to selected target organs. 相似文献
9.
Stabilization of alpha-chymotrypsin (CT) by covalent immobilization on the amine-functionalized magnetic nanogel was studied. The amino groups containing superparamagnetic nanogel was obtained by Hoffman degradation of the polyacrylamide (PAM)-coated Fe(3)O(4) nanoparticles prepared by facile photochemical in situ polymerization. CT was then covalently bound to the magnetic nanogel with reactive amino groups by using 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide as coupling reagent. The binding capacity was determined to be 61mg enzyme/g nanogel by BCA protein assay. Specific activity of the immobilized CT was measured to be 0.93U/(mgmin), 59.3% as that of free CT. The obtained immobilized enzyme had better resistance to temperature and pH inactivation in comparison to free enzyme and thus widened the ranges of reaction pH and temperature. The immobilized enzyme exhibited good thermostability, storage stability and reusability. Kinetic parameters were determined for both the immobilized and free enzyme. The value of K(m) of the immobilized enzyme was larger than did the free form, whereas the V(max) was smaller for the immobilized enzyme. 相似文献
10.
Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein (P3) that is indispensable for viral infectivity. Although P3 has been shown to be a prerequisite for CaMV aphid transmission, its role in viral replication remains unknown. We previously showed that P3 forms a tetramer in planta and that P3 tetramer co-sediments with viral coat protein on sucrose gradient centrifugation, suggesting that a tetramer may be the functional form of P3. We presumed that disulfide bonds were involved in tetramer formation because 1) the tetramer was detected by Western blotting after electrophoresis under non-reducing conditions, and 2) the cysteine-X-cysteine motif is well conserved in CaMV P3 and P3 homologues among Caulimoviruses. Therefore we mutated either or both of the cysteine residues of CaMV P3. The mutant viruses were infectious and accumulated to a similar extent as the wild-type. An analysis of mutant proteins confirmed that the wild-type P3 molecules in the tetramer are covalently bound with one another through disulfide bonds. It was also suggested that mutant proteins are less stable than wild-type protein in planta. Furthermore, sedimentation study suggested that the disulfide bonds are involved in stable association of P3 with CaMV virions or virion-like particles, or both. The mutant viruses could be transmitted by aphids. These results suggested that the covalent bonds in P3 tetramer are dispensable for biological activity of P3 under experimental situations and may have some biological significance in natural infection in the field. 相似文献
11.
Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b. 总被引:8,自引:1,他引:7
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Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [3H]palmitate or [3H]palmitate and [14C]ribose followed by chloroform-methanol extraction could most of the 3H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP. 相似文献
12.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,886(1):64-75
We have tested the feasibility of targeting liposomes via interaction with specific ecto-enzymes, i.e., enzymes which have their active site oriented to the external surface of the cell. 3,4-Dimethylpyridine adenine dinucleotide, a competitive inhibitor of ecto-NAD+-glycohydrolase, was substituted at N6 with a hydrophilic spacer arm, functionalized with a sulfhydryl group, and covalently linked to preformed liposomes containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. We show that compared to control vesicles, the binding of the conjugated liposomes was greatly increased (up to 5-fold) to cells presenting ecto-NAD+-glycohydrolase activity (Swiss 3T3 fibroblasts, mouse peritoneal macrophages); in contrast, no specific binding was detected with hepatoma tissue culture cells, which lack this enzyme. Specific binding was found to depend on the ligand/lipid molar ratio of the vesicles and on the length of the arm. High concentrations of free 3,4-dimethylpyridine adenine dinucleotide virtually abolished the specific binding to cells of the targeted liposomes. Analysis of binding revealed that the ligand conjugated to the liposomes presented a functional affinity for 3T3 fibroblasts 15-fold superior to that of the free ligand. 相似文献
13.
Raw-starch-digesting enzyme (RSDA) was immobilized on Amberlite beads by conjugation of glutaraldehyde/ polyglutaraldehyde (PG)-activated beads or by crosslinking. The effect of immobilization on enzyme stability and catalytic efficiency was evaluated. Immobilization conditions greatly influenced the immobilization efficiency. Optimum pH values shifted from pH 5 to 6 for spontaneous crosslinking and sequential crosslinking, to pH 6-8 for RSDA covalently attached on polyglutaraldehyde-activated Amberlite beads, and to pH 7 for RSDA on glutaraldehyde-activated Amberlite. RSDA on glutaraldehyde-activated Amberlite beads had no loss of activity after 2 h storage at pH 9; enzyme on PG-activated beads lost 9%, whereas soluble enzyme lost 65% of its initial activity. Soluble enzyme lost 50% initial activity after 3 h incubation at 60 degrees C, whereas glutaraldehyde-activated derivative lost only 7.7% initial activity. RSDA derivatives retained over 90% activity after 10 batch reuse at 40 degrees C. The apparent Km of the enzyme reduced from 0.35 mg/ml to 0.32 mg/ml for RSDA on glutaraldehyde-activated RSDA but increased to 0.42 mg/ml for the PG-activated RSDA derivative. Covalent immobilization on glutaraldehyde Amberlite beads was most stable and promises to address the instability and contamination issues that impede the industrial use of RSDAs. Moreover, the cheap, porous, and non-toxic nature of Amberlite, ease of immobilization, and high yield make it more interesting for the immobilization of this enzyme. 相似文献
14.
《生物化学与生物物理学报:生物膜》1985,814(1):151-155
A method is described for the preparation of liposomes carrying covalently attached protein at the outer surface. It is based upon the reaction between proteins, thiolated with the heterobifunctional reagent succinimidyl-S-acetylthioacetate (SATA), and liposomal maleimido-4-(p-phenylbutyryl)phosphatidyl-ethanolamine. Advantages of the procedure are that it is not restricted to proteins carrying native SH-groups and that it is substantially more convenient and less time-consuming than previously published methods. Applications for these liposomes are to be found in the fields of liposome targeting and the production of monoclonal antibodies. 相似文献
15.
We describe a process for covalently linking proteins to glass microscope slides and microbeads in a manner that optimizes the reactivity of the immobilized proteins and that is suitable for high-throughput microarray and flow cytometry analysis. The method involves the diazo coupling of proteins onto activated self-assembled monolayers formed from p-aminophenyl trimethoxysilane. Proteins immobilized by this method maintained bioactivity and produced enhanced levels of protein-protein interaction, low background fluorescence, and high selectivity. The binding of immobilized proteins to their specific binding partner was analyzed quantitatively and successfully correlated with solution concentrations. Diazotized surfaces bound more efficiently to proteins containing a hexahistidine tag than those without a his-tag. Moreover, significantly higher reactivity of the immobilized his-tagged proteins was observed on diazotized surfaces than on amine-terminated surfaces. Results suggest that his-tagged proteins are immobilized by reaction of the his-tag with the diazotized surface, thus offering the possibility for preferential orientation of covalently bound proteins. 相似文献
16.
Bolivar JM Wilson L Ferrarotti SA Fernandez-Lafuente R Guisan JM Mateo C 《Biomacromolecules》2006,7(3):669-673
Formate dehydrogenase (FDH) is a stable enzyme that may be readily inactivated by the interaction with hydrophobic interfaces (e.g., due to strong stirring). This may be avoided by immobilizing the enzyme on a porous support by any technique. Thus, even if the enzyme is going to be used in an ultra-membrane reactor, the immobilization presents some advantages. Immobilization on supports activated with bromocianogen, polyethylenimine, glutaraldehyde, etc., did not promote any stabilization of the enzyme under thermal inactivation. However, the immobilization of FDH on highly activated glyoxyl agarose has permitted increasing the enzyme stability against any distorting agent: pH, T, organic solvent, etc. The time of support-enzyme reaction, the temperature of immobilization, and the activation of the support need to be optimized to get the optimal stability-activity properties. Optimized biocatalyst retained 50% of the offered activity and became 50 times more stable at high temperature and neutral pH. Moreover, the quaternary structure of this dimeric enzyme becomes stabilized by immobilization under optimized conditions. Thus, at acidic pH (conditions where the subunit dissociation is the first step in the enzyme inactivation), the immobilization of both subunits of the enzyme on glyoxyl-agarose has allowed the enzyme to be stabilized by hundreds of times. Moreover, the optimal temperature of the enzyme has been increased (even by 10 degrees C at pH 4.5). Very interestingly, the activity with NAD(+)-dextran was around 60% of that observed with free cofactor. 相似文献
17.
Mice were immunized with purifiedCryptococcus neoformans soluble polysaccharide, or with the polysaccharide coupled to methylated bovine serum albumin or methylated bovine gamma globulin. The polysaccharide-methylated protein complexes were no more immunogenic than the purified polysaccharide when used without Freund's incomplete adjuvant; however, the methylated protein complexes induced higher levels of antibody than purified polysaccharide when emulsified in the adjuvant. Sera from mice were titrated by passive hemagglutination, and maximum antibody titers were observed 14 to 21 days after immunization. Antibody titers declined rapidly after 14 to 21 days in mice immunized with antigen alone; whereas, animals immunized with cryptococcal antigen emulsified in adjuvant remained at peak levels throughout a six week experimental period. All antigens were immunogenic over a wider dosage range when contained in adjuvant. Individual mice immunized with an adjuvant emulsion of purified polysaccharide varied widely in the amount of antibody produced, with some of the animals producing no detectable antibody. 相似文献
18.
The effect of tentoxin on the binding of adenine nucleotides to soluble chloroplast coupling factor (CF1) has been studied and the following results have been obtained: 1. Tentoxin (400 micron) increases the maximum attainable tight binding of ADP to CF1. In the absence of tentoxin, the maximal binding observed by the method employed is about 0.3 nmol ADP/mg protein, whereas in the presence of tentoxin this ranges from 1.5 to 2.0 nmol ADP/mg protein. 2. Tentoxin-induced binding of ADP to CF1 is severely inhibited by divalent cations (50% inhibition at about 2 mM) but only weakly inhibited by monovalent cations (less than 50% inhibition at 100 mM). 3. The binding of ADP to CF1 induced by tentoxin is inhibited by ATP and adenylyl imidodiphosphate but is not inhibited by other nucleotides including AMP, GDP, CDP, IDP, or beta, gamma-methylene ATP. 4. The ADP-CF1 complex induced by tentoxin is quite stable. 75% remains bound to CF1 even after passage of the complex through a gel filtration column. An additional 25% can be removed by incubation in the presence of ADP, and all of the bound ADP can be removed only after incubation in the presence of both tentoxin and ADP. The latter result is interpreted as a tentoxin-induced exchange of bound ADP for medium ADP. 相似文献
19.
Oxidation of linoleic acid encapsulated with soluble soybean polysaccharide by spray-drying 总被引:2,自引:0,他引:2
Minemoto Y Fang X Hakamata K Watanabe Y Adachi S Kometani T Matsuno R 《Bioscience, biotechnology, and biochemistry》2002,66(9):1829-1834
Linoleic acid was encapsulated with a soluble soybean polysaccharide, gum arabic, or a mixture of both together with maltodextrin, and the oxidation process of the encapsulated acid was measured at 37 degrees C and at a relative humidity of 12%. The soybean polysaccharide was more effective for encapsulating the acid and suppressing the oxidation of the encapsulated acid than gum arabic. A mixture of the soybean polysaccharide and maltodextrin was also effective for this purpose when the weight fraction of the polysaccharide was equal to or greater than 0.75. 相似文献
20.
We have investigated methods of stabilizing prolidase by chemical modification and covalent coupling to various supports, for use in protein hydrolysis and possible use in enzyme replacement therapy. Purified acetone powder of calf brain prolidase was further purified by gel filtration on Sephadex G-200 and chromatography on DEAE-Sephadex A25. Polyacrylamide gel electrophoresis showed that the number of bands was reduced from 11 to 2. Since yields were low, the purified (NH4)2SO4 fraction was used in all experiments. Thiolation of the enzyme reduced the amount of protein coupled to AH-or CH-Sepharose 4B. Activities were highest when the protein was linked through its carboxyl groups. The coupled enzyme showed much greater thermal stability than its free counterpart. Of the bound preparations, the thiolated was less stable than the untreated. Untreated and thiolated enzymes bound to either matrix showed higher activity at low pH and less at high pH than the free material. Thiolation shifted the pH maximum from 6.8 to 7.5. The free thiolated enzyme and that bound to activated SH-Sepharose 4B showed greater thermal stability and a broader pH range of optimal activity than the bound untreated enzyme. These results show that prolidase can be immobilized by coupling to an insoluble matrix through various types of covalent bonds with retention of activity and increased stability. 相似文献