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1.
Summary. The capability of ficin, a cystine protease, to form peptide bonds was investigated using several types of N-Boc-amino acid phenyl and naphthyl esters as acyl donor components. Enzyme-catalyzed peptide synthesis was carried out under optimized reaction conditions of pH, acyl acceptor concentration and selection of the best yield organic solvent. It used a condensation of N-Boc-Ala-OpGu and Ala-pNA as a model reaction. The products were obtained in 72–96% yield using 10 different substrates, within a few minutes of reaction time. Authors’ address: Prof. Haruo Sekizaki, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan  相似文献   

2.
Oxovanadium (IV)-Schiff-base complexes and those bound on Merrifield resin as a polymer support were prepared for their characterization and their use as catalyst in oxidation of methyl phenyl sulfide. Schiff bases were prepared with use of salicylaldehyde and 2,4-dihydroxybenzaldehyde as the aldehydes and 2-aminoethanol, L-phenylalaninol, L-histidinol, and L-phenylalanine as the counterparts. Oxovanadium (IV) complexes made up of these Schiff bases and those bound on the resin were spectroscopically characterized. The polymer-supported Schiff-base complexes in the presence of tertiary-butylhydroperoxide converted the sulfide to the corresponding sulfoxide in 80-90% yield in CDCl3 in 90 min. They afforded slightly lower rates of oxidation than the corresponding monomeric complexes. They converted the sulfide in a stereoselective manner yielding the sulfoxide in enantiomeric excess (the highest value of 40%). The polymer-supported complexes and the corresponding monomers achieved almost the same enatiometric excesses with each other.  相似文献   

3.
The aim of the study was to demonstrate the applicability of differential scanning calorimetry (DSC) on porosity analysis for cellulose and starch. Croscarmellose sodium (CCS) and sodium starch glycolate (SSG) were allowed to sorb moisture in 85%, 90%, 95%, and 100% relative humidity (RH) at 40°C for 24 hours. The pretreated samples were then subjected to DSC running temperature ranging from 25°C to −50°C at a cooling rate of 10°C/min. The cooling traces of water crystallization, if present, were transformed to porosity distribution via capillary condensation using Kelvin's equation. The porosity analysis of CCS and SSG was also done using nitrogen adsorption as a reference method. It was found that sorbed water could not be frozen (in cases of 85% and 90% RH) until the moisture content exceeded a cutoff value (in cases of 95% and 100% RH). The nonfreezable moisture content was referred to tightly bound, plasticizing water, whereas the frozen one may be attributed to loosely bound water condensation in pore structure of CCS and SSG surfaces. Not only capillary condensation but also the tightly bound, nonfreezable monolayer water lying along the inner pores of the surface contributed to porosity determination. Good agreement with less than 5% deviation of mean pore size was observed when the results were compared with nitrogen adsorption. The narrower pore size distributions, however, were obtained because of the limitations of the technique. It was concluded that pore analysis by DSC could be successful. Further research needs to be done to account for limitations and to extend the applicability of the technique.  相似文献   

4.
N-Methyl-alpha,alpha-diphenyl-L-prolinol derivatives with para-bromo substituents in one or both of the phenyl rings are easily bound to crosslinked polystyrene beads containing phenylboronic acid residues using Suzuki reactions. When the products were used as catalysts for the reactions of aldehydes with diethylzinc in toluene at 20 degrees C, the alcohols were produced in chemical yields >90% and with ees of upto 94%. The better of the two supported catalysts gave ees only 0-9% lower than those obtained with the corresponding soluble catalyst. One of the supported catalysts was recycled successfully nine times.  相似文献   

5.
Washed-cell preparations of recombinant Escherichia coli JM109(pDTG141), engineered to express the naphthalene dioxygenase (NDO) gene from Pseudomonas sp. NCIB 9816-4, have been used to biooxidise a range of aryl alkyl-, dialkyl- and bicyclic sulfides. A series of 16 phenyl alkyl sulfides was oxidised to equivalent sulfoxides, typically with moderate to high (>90%) yield and high enantioselectivity (>85% ee), the (S)-enantiomer being the predominant product, with little if any further oxidation. The addition of some electron-donating or electron-withdrawing groups to the phenyl ring decreased yield and/or stereoselectivity of the NDO-catalysed biotransformation, whereas increasing the size of the alkyl chain (nC3H7, iC3H7 and nC4H9) resulted in a notable inversion in selectivity to yield (R)-series sulfoxides (>74% ee) as the predominant products. The addition of one or more methylene groups between the phenyl ring and sulfur atom resulted in notable reductions in both the yield and stereoselectivity of the (S)-predominant sulfoxidations. With the exception of cyclohexyl- and n-hexyl methyl sulfide which both gave (S)-sulfoxides with good stereoselectivity and yield, other dialkyl- and bicyclic sulfides were poor substrates for sulfoxidation by NDO. Both the close agreement with data obtained using purified NDO and the absence of stereoselective sulfoxidation in equivalent controls with the E. coli JM109 host support the contribution made by the cloned NDO carried on the pDTG141 plasmid.  相似文献   

6.
The total sequential solid phase synthesis of AcA98STnC(90-123)amide is described. The yield is comparable to that obtained previously using fragment condensation on the solid support. HPLC purification results in a product which displays greater calcium induced conformational changes than the peptide prepared by the fragment condensation method. However, the former has a dissociation constant for calcium (28 microM) seven times higher than the latter (4 microM).  相似文献   

7.
The aim of the work was to study the properties of the bacterial cellulose membrane (BCM) and the feasibility of using it as a new, environmentally friendly support carrier for yeast cell immobilization. It was observed that the morphology of BCM varied with different cultivation methods and the scanning electron microscopy (SEM) images confirmed that the yeast cells were entrapped in the porous network of BCM obtained from the static culture and stabilized by the cross-linked fibrils. Particularly, the research confirmed the effectiveness of yeast immobilization in BCM reflected by the high yield of alcohol (9.7% v/v, a 21.25% increase of those using free cells) and the high stability. The specific rate of ethanol production by the immobilized cells in BCM was 2.1 g g−1 h−1, 31.3% greater than that of the suspended cells. Results implied that applying BCM as the support carrier had little adverse effects on cell viability and proliferation. Instead, it facilitated the product leakage and nutrients transportation through the porous network.  相似文献   

8.
酶固定化的新型载体──PF凝胶应用研究   总被引:3,自引:0,他引:3  
用对苯二酚和甲醛在酸催化下制得新一类凝胶,此凝胶价廉,易于制备,多孔、无毒、亲水性强;能选择性吸附多糖,而用于多糖与低聚糖及单糖的分离;可作为载体对多种酶及蛋白质给予固定,与蛋白质的最大结合量为558mg/g,固定糖化酶时的活力回收达84%;此固化酶对淀粉的转化率达93%,当加入一定量的间苯二酚,可得改性的PF凝胶,其性能又有提高,可见PF凝胶为新型的酶的固定化优良载体.  相似文献   

9.
Digestive enzymes from human intestinal mucosa were solubilized by Triton X-100 and papain and covalently bound to eyanogen bromide-activated Sepharose 4-B gel. Triton X-100 solubilized most of the activities, and 39.1 to 63.5% were immobilized on the carrier. The other enzymes, still bound on the microvilli, were subsequently solubilized by papain but then the yield of immobilization reached only 11.0 to 17.6%. The enzyme-Sepharose gel was freeze-dried with a filler and stored without loss of activity. The rate of hydrolysis of di- and trisaccharides, dipeptides, and p-nitrophenylphosphate was measured by incubation on a small column containing less than 0.03 U of immobilized activities. The enzymatic multiplicity and catalytic properties of the intestinal mucosa enzymes were fully recovered on the carrier. This method is proposed for routine evaluation of the digestibility of dipeptides and synthetic disaccharides.  相似文献   

10.
Subtilisin Carlsberg (SC) was shown to catalyze the solid phase segment coupling of peptides in complex with sodium dodecyl sulfate (SDS) in an organic medium on Aminosilochrom and polyvinyl alcohol (PVA) cryogel activated with glutaraldehyde or divinylsulfone. Diamines of different lengths with a general formula NH2-(CH2) n -NH2 (n = 2, 4, and 6) were used as spacers between the PVA cryogel and the peptide. A model reaction of enzymatic attachment of the Dnp-Ala-Ala-Leu-OMe tripeptide to the PVA cryogel was carried out by treatment with the SDS-SC complex in a mixture of anhydrous ethanol and DMSO (7 : 3, v/v) using a tenfold excess of the carboxyl component. The molar enzyme-substrate ratio was 1 : 88. The effect of the method of matrix activation, length of a spacer, and reaction time on the coupling efficiency was studied. Hexamethylenediamine was found to be the most effective spacer for the enzymatic coupling on the PVA cryogel activated with glutaraldehyde (the reaction proceeded with the highest yield of 60%). The reaction efficiency was considerably lower in the case of ethylenediamine and tetramethylenediamine (10 and 15%, respectively). The best results were obtained on the PVA cryogel activated by divinylsulfone with hexamethylenediamine as a spacer. A two-step condensation of tripeptides was carried out on this support. The second step of condensation was shown to proceed better (in 85% yield) in comparison with the first step (37% yield).  相似文献   

11.
The synthesis of a [Glu5, Ala12, Ala18,-Ala21]sheep insulin-A-chain by condensation of 5 fragments on a polymer support is described. The 5 fragments Boc-Gly-Ile-Val-Glu(gammaOBut)-Glu(gammaOBut) (V), Boc-Cys(SiPr) (IV), Boc--Cys(SiPr)-Ala-Gly-Val (III), Boc-Cys(SiPr)-Ala-Leu-Tyr-Gln-Leu (II) and Boc-Glu(gammaOBut)-Ala--Tyr(Bzl)-Cys(SiPr)-N2H3 (I) were synthesized by conventional methods and coupled with dicyclohexylcarbodiimide/1-hydroxybenzotriazol (II, III, IV, V) and by the azide method (I) with coupling yields of 60-98% on an Ala-polymer. The failure sequence peptides were separated by ion exchange chromatography on DEAE-Sephadex and by chromatography on Biogel P4. The A-chain was obtained in 9% yield, which, after combinations with natural B-chain, gave insulin activities comparable to that obtained with natural A-chain. These results demonstrate that fragment condensation by the solid-phase method together with simple techniques for purification can be used for the synthesis of longer peptides.  相似文献   

12.
The objective of this study was to test a new model for the homodimeric animal FAS which implies that the condensation reaction can be catalyzed by the amino-terminal beta-ketoacyl synthase domain in cooperation with the penultimate carboxyl-terminal acyl carrier protein domain of either subunit. Treatment of animal fatty acid synthase dimers with dibromopropanone generates three new molecular species with decreased electrophoretic mobilities; none of these species are formed by fatty acid synthase mutant dimers lacking either the active-site cysteine of the beta-ketoacyl synthase domain (C161A) or the phosphopantetheine thiol of the acyl carrier protein domain (S2151A). A double affinity-labeling strategy was used to isolate dimers that carried one or both mutations on one or both subunits; the heterodimers were treated with dibromopropanone and analyzed by a combination of sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Western blotting, gel filtration, and matrix-assisted laser desorption mass spectrometry. Thus the two slowest moving of these species, which accounted for 45 and 15% of the total, were identified as doubly and singly cross-linked dimers, respectively, whereas the fastest moving species, which accounted for 35% of the total, was identified as originating from internally cross-linked subunits. These results show that the two polypeptides of the fatty acid synthase are oriented such that head-to-tail contacts are formed both between and within subunits, and provide the first structural evidence in support of the new model.  相似文献   

13.
Uhlik O  Kamlar M  Kohout L  Jezek R  Harmatha J  Macek T 《Steroids》2008,73(14):1433-1440
The aim of this work was to isolate plant ecdysteroid-binding proteins using affinity chromatography. Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown although ecdysteroids occur in some plants in a relatively large amount. Therefore, 20-hydroxyecdysone was immobilized on a polymeric carrier as a ligand for affinity chromatography in order to isolate plant ecdysteroid-binding proteins from the cytosolic extract of New Zealand spinach (Tetragonia tetragonoides). Non-specifically bound proteins were eluted with a rising gradient of concentration of sodium chloride, and 3% (v/v) acetic acid was used for the elution of the specifically bound proteins. Using this method, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was isolated. The influence of ecdysteroids on RuBisCO was further studied. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO(2) is fixed into organic matter by more than 10%.  相似文献   

14.
The synthesis of a series of SS'-polymethylenebis(methanethiosulphonates) including the pentane, hexane, octane, decane and dodecane derivatives is described. These derivatives were synthesized by condensation between dibromoalkanes and potassium methanethiosulphonate in refluxing methanol and this seems an especially versatile reaction for the synthesis of asymmetric thiosulphonate derivatives. The synthesis of SS'-[1,8-3H4]-octamethylenebis(methanethiosulphonate) was also perfomed. Cross-linking was demonstrated in the four enzymes lactate dehydrogenase, phosphofructokinase, pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase. For all four enzymes cross-linking was efficiently reversed by reducing conditions in denaturing solvents. The reaction with glyceraldehyde 3-phosphate dehydrogenase was unique in that only the cross-linked dimer was produced in significant amounts (greater than 90% of total products as dimer). This reaction was followed in detail with radioactive cross-linking reagent. Inhibition of enzyme activity was extremely fast and showed an asymmetric distribution of enzyme activity on subunits. Thus complete modification of only one subunit resulted in up to 75% inhibition of enzyme activity. Reaction of glyceraldehyde 3-phosphate dehydrogenase with 1.25 mol of SS'-octamethylenebis(methanethiosulphonate) per mol of enzyme subunit produced two species of protein. The first species was obtained in 20% yield and was only partially re-activated on mild reduction with 2-mercaptoethanol. The second species was isolated in 66% yield and was completely re-activated on mild reduction. Before reduction there was 4 mol of inhibitor per tetramer for the latter species, and more than 95% of the enzyme was present as a dimer on non-reducing electrophoresis. After mild reduction 2 mol of inhibitor was still bound per tetramer, the enzyme was now catalytically active and the dimer was still the major structure on non-reducing electrophoresis. Thus mild reduction of SS'-octamethylenebis(methanethiosulphonate-treated glyceraldehyde 3-phosphate dehydrogenase enabled the production of active enzyme in which there is a stable cross-link across one of the molecular axes of the tetrameric enzyme. This cross-link was only reversed if reduction was performed when the enzyme was denatured. The molecular weight of cross-linked and re-activated cross-linked glyceraldehyde 3-phosphate dehydrogenase was established as 144000 (tetramer) by sucrose-density-gradient centrifugation. These observations are interpreted in terms of the molecular structure of glyceraldehyde 3-phosphate dehydrogenase.  相似文献   

15.
Aspartate aminotransferase (L-aspartate : 2-oxoglutarate aminotransferase, EC 2.6.1.1) has been covalently bound to chemically activated collagen films. This enzyme had never previously been coupled to any other solid support. The coupling method, including acyl azide formation on the carrier, allowed coupling of many other enzymes. A systematic study of coupling conditions has been performed; influence of time of coupling and of concentration of coupling solution on the enzymatic activity retained on the film. Coupling solutions could be used for several successive couplings. To determine the yield of binding, N-[14C] ethylmaleimide-labelled enzyme was prepared fully active and bound to collagen films. After lyophilisation the film retained most of its activity when stored in buffer and the half-life of the enzymatic film was about ten months. pH Dependence and activation energy were about the same for soluble and coupled enzyme. Coupling protects against thermal denaturation and increases the stability of the enzyme; the enzymatic film could be used repeatedly. Kinetics were somewhat modified in the coupled enzyme as compared to the enzyme in solution. Glutamate appeared more available while oxaloacetate seemed to be limiting. These modifications might be due to the proteic support itself. The enzymatic films also revealed themselves as a good tool for industrial or clinical purposes as well as for studying the mechanism of enzyme action.  相似文献   

16.
AIMS: Anaerobic fungi are important members of the fibrolytic community of the rumen. The aim of this study was to study their requirement for aromatic amino acids (AA) and related phenyl acids (phenylpropionic and phenylacetic acids) for optimal xylan fermentation. METHODS AND RESULTS: Neocallimastix frontalis RE1 and Piromyces communis P were grown in a defined medium containing oat spelts xylan as the sole energy source, plus one of the following N sources: ammonia; ammonia plus a complete mixture of 20 AA commonly found in protein; ammonia plus complete AA mixture minus aromatic AA; ammonia plus phenyl acids; ammonia plus complete AA mixture without aromatic AA plus phenyl acids. Both species grew in all the media, indicating no absolute requirement for AA. The complete AA mixture increased (P<0.05) acetate concentration by 18% and 15%, sugar utilization by 33% and 22% and microbial yield by about 22% and 15% in N. frontalis and P. communis, respectively, in comparison with the treatments that had ammonia as the only N source. Neither the supply of aromatic AA or phenol acids, nor their deletion from the complete AA mixture, affected the fermentation rate, products or yield of either species. CONCLUSIONS: AA were not essential for N. frontalis and P. communis, but their growth on xylan was stimulated. The effects could not be explained in terms of aromatic AA alone. SIGNIFICANCE AND IMPACT OF THE STUDY: Ruminant diets should contain sufficient protein to sustain optimal fibre digestion by ruminal fungi. Aromatic AA or phenyl acids alone cannot replace the complete AA mixture.  相似文献   

17.
Deuterium NMR spectra were obtained for L-phenylalanine-d5, deuterated on the phenyl ring, in cross-linked polycrystalline samples of carboxypeptidase A containing different amounts of water. The deuterium powder pattern line shapes are simulated by extension of the theory to include both a local reorientational motion of the bound L-phenylalanine phenyl ring and exchange of the L-phenylalanine with an intracrystalline isotropic environment. The spectral simulations are consistent with the phenyl ring of the phenylalanine executing pi-flips in the bound environment at rates that vary from 3 x 10(4) Hz at 6% water content to 1 x 10(5) Hz at 21% water content. At all water contents studied, the ligand exchanges with an essentially isotropic environment in the crystal with a rate constant of approximately 2.5 x 10(-3) Hz. Although the dissociation constant for the L-phenylalanine is only 18 mM, the spectral simulations that reproduce the experimental line shape well do not require significant wobble of the phenyl ring rotation axis, which is consistent with the binding interactions identified by x-ray crystallography.  相似文献   

18.
Synthesis of a membrane protein with two transmembrane regions.   总被引:1,自引:0,他引:1  
A membrane protein with two transmembrane domains was synthesized by means of the thioester method. The F1F0 ATP synthase subunit c (Sub.c), which consists of 79 amino acid residues (MW 8257), was chosen as a target. For synthetic purposes, two building blocks, Boc-[Lys34(Boc)]-Sub.c(1-38)-SCH2CH2CO-Ala and Sub.c(39-79), were synthesized via solid-phase methods using Boc chemistry. RP-HPLC purification conditions for the transmembrane peptide were examined. As a result, a combination of a mixture of formic acid, 1-propanol and water with a phenyl column was found to be useful for separating the transmembrane peptide. The purified building blocks were condensed in DMSO in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), N,N-diisopropylethylamine to give the product, Sub.c, after removal of Boc groups (yield 16%). The yield of the condensation reaction could be improved to 23% by raising the reaction temperature to 50 degrees C, and to 26% when a mixture of chloroform and methanol was used as a solvent.  相似文献   

19.
A palmitoyl CoA-protein complex was isolated from bovine heart mitochondria and purified to homogeneity. The elution profile of the [14C]palmitoyl CoA bound protein from a hydroxyapatite column was identical to that seen when [3H]carboxyatractylate was used as the bound ligand. A sample of the palmitoyl CoA-protein complex from a peak fraction of the column appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. The mobility of the protein bound with palmitoyl CoA was identical to the one bound with carboxyatractylate and the molecular weight was estimated to be 30,000 daltons. Compared to the stable palmitoyl CoA-protein complex, purification of the unliganded carrier from mitochondria at 22°C resulted in a disaggregated protein. These physical characteristics of the palmitoyl CoA-protein complex correspond to those identified for the ADPATP carrier. The results further confirm the specificity of the fatty acyl CoA ligand for the adenine nucleotide translocase and support the concept that it may be a physiological modulator of adenine nucleotide translocation.  相似文献   

20.
Alumina-doped alginate gel (AEC) was developed as a new type of cell carrier to be used in ethanol fermentation. The presence of the alumina particles in alginate gel not only improved the porous structure of the carrier, but also provided many advantageous characteristics including good mechanical strength, high stability, and high immobilization yield. The attachment of alumina particles and yeast cells by electrostatic attraction was shown to promote cell growth and increase ethanol productivity. The AEC carrier was found to be more effective for the immobilization of Saccharomyces cerevisiae M30 than the conventional Ca-alginate bead. Ethanol productivities of 1.4 and 7.9 ∼ 12.6 g/(L/h) were obtained using the AEC cultures in batch and continuous modes of operation, respectively, with an ethanol yield of 43.9 ∼ 46.7% and an immobilized yield of 81.4 ∼ 84.5%. Ethanol fermentation in a continuous packed-bed reactor using the AEC carrier was stable for > 30 days.  相似文献   

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