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1.
The capability of Glu/Asp-specific endopeptidase from Bacillus licheniformis to form Glu/Asp-Xaa bonds in frozen aqueous systems was investigated. Under frozen state conditions, the enzyme was able to catalyse peptide bond formation more effectively than in liquid reaction mixtures. The acceptance of amino components which were completely inefficient nucleophiles at room temperature indicates a changed specificity of Glu/Asp-specific endopeptidase under frozen state conditions. Protease-catalysed coupling of two acidic amino acids was demonstrated for the first time. The utilization of Glu/Asp-specific endopeptidase from Bacillus licheniformis in frozen aqueous systems offers new possibilities in enzyme-catalysed peptide synthesis.  相似文献   

2.
During the past decade proteases have been widely used as catalysts in peptide synthesis. Unfortunately, they are not ideal ligases. Enzymatic peptide synthesis in frozen aqueous systems has been developed as an approach towards the suppression of competitive reactions. This paper summarizes reports concerning the behaviour of non-enzymatic as well as of enzyme-catalysed reactions when the reaction mixture is frozen. The advantages of freezing the reaction mixture in serine and cysteine protease-catalysed peptide synthesis, the influence of modified reaction conditions and the possible reasons for the yield-increasing effect of freezing are discussed.  相似文献   

3.
The continuous alpha-chymotrypsin-catalyzed peptide synthesis of kyotorphin, tyrosyl-arginine, via the N(alpha) formyltyrosyl-arginine propyl ester is described. For continuous process development, two reaction systems were studied: immobilized alpha-chymotrypsin covalently bound to Eupergit C packed in a column, and soluble alpha-chymotrypsin utilizing an enzyme membrane reactor. Selectivities and kinetic parameters are discussed. The use of soluble enzyme in an enzyme membrane reactor proved superior to the covalently immobilized enzyme. A significant loss of enzyme activity and a certain decrease of selectivity was observed during immobilization. It was shown that the addition of organic solvent, in this case n-propanol, causes a severe diminuation of the enzyme activity.  相似文献   

4.
C D Tormanen 《Cryobiology》1992,29(4):511-518
Polyethylene glycol is a water-soluble polymer which is widely used in the pharmaceutical, cosmetic, and chemical industries. In this study, it is shown that polyethylene glycol is an effective cryoprotectant of rat kidney transamidinase purified from both the mitochondria and cytosol. Much of the activity is lost when the purified enzyme is frozen and thawed in sodium-potassium phosphate buffer in the absence of cryoprotectants. Polyethylene glycols with molecular weights of 4000 to 10,000 were effective cryoprotectants. However, polyethylene glycols with a molecular weight of 1000 or lower inhibited the purified enzyme. A concentration of only 0.01% polyethylene glycol 4000, 8000, or 10,000 was required for complete cryoprotection. In addition to polyethylene glycol, 0.5 mM ethylenediaminetetraacetic acid was required in the phosphate buffer for complete cryoprotection. The stabilization of purified transamidinase by polyethylene glycol will facilitate characterization experiments designed to compare the properties of the mitochondrial and cytosolic isozymes.  相似文献   

5.
The object of this research is to study acoustic emissions (AE) in aqueous solutions of polyethylene glycols (PEGs) of molecular masses from 300 to 3000 during cooling at 100 degrees C/min and heating at 70 degrees C/min in the temperature range from 0 to -196 degrees C. The dependence of AE intensity on PEG concentration and molecular mass is analysed. The AE intensity correlated with the crystalline to amorphous phase ratio in the frozen system.  相似文献   

6.
Bacterial Utilization of Ether Glycols   总被引:9,自引:8,他引:1       下载免费PDF全文
A soil bacterium capable of using oligo- and polyethylene glycols and ether alcohols as sole sources of carbon for aerobic growth was isolated. The effects of substituent groups added to the ether bonds on the acceptability of the compounds as substrates were studied. Mechanisms for the incorporation of two-carbon compounds were demonstrated by the observation that acetate, glyoxylate, ethylene glycol, and a number of the tricarboxylic acid cycle intermediates served as growth substrates in minimal media. The rate of oxidation of the short-chained ethylene glycols by adapted resting cells varied directly with increasing numbers of two-carbon units in the chains from one to four. The amount of oxygen consumed per carbon atom of oligo- and polyethylene glycols was 100% of theoretical, but only 67% of theoretical for ethylene glycol. Resting cells oxidized oligo- and polyethylene glycols with 2 to 600 two-carbon units in the chains. Longer chained polyethylene glycols (up to 6,000) were oxidized at a very slow rate by these cells. Dehydrogenation of triethylene glycol by adapted cells was observed, coupling the reaction with methylene blue reduction.  相似文献   

7.
This is a comparative study of the performance of thermolysin for enzymatic peptide synthesis by reversed hydrolysis in several different reaction systems. Z-Gln-Leu-NH(2) was synthesized in acetonitrile containing 5% water (with various catalyst preparation methods) as well as by the "solid-to-solid" and frozen aqueous methods. Reaction rates (values in nanomoles per minute per milligram) in acetonitrile depended significantly on the method of addition of enzyme: (a) direct suspension in the reaction mixture as freeze-dried powders gave 60 to 95; (b) addition as an aqueous solution, so that enzyme precipitates on mixing with acetonitrile, gave 230; (c) addition as an aqueous suspension gave a remarkable increase in reaction rates (up to 780); (d) immobilized enzymes (adsorbed at saturating loading on celite, silica, Amberlite XAD-7, or polypropylene, then dried by propanol rinsing) all gave <230. It is postulated that, starting with the enzyme already in the form of solid particles in aqueous buffer, there is a minimum chance of alteration of its optimal conformation during transfer to the organic medium. For solid-to-solid synthesis with 10% water content we found initial rates of 670 under optimized conditions. In frozen aqueous synthesis, rates were <10. Equilibrium yields were always around 60% in low water organic solvent, whereas they were found to >80% in the aqueous systems studied.  相似文献   

8.
PPL-catalyzed synthesis of the precursor dipeptides of RGD as a cellular adhesion factor, Benzyl-Arg-Gly-NH2 and CBZ-Gly-Asp-NH2, was conducted in water-organic cosolvents systems. Five water-miscible organic solvents, which have some advantage over the water-immiscible organic solvent systems or the anhydrous organic solvent systems used often in protease-catalyzed synthesis of a peptide bond, were tested. The reaction condition of PPL-catalyzed synthesis of the dipeptides was optimized by examining the main factors affecting the product yield. The optimal reaction condition for the synthesis of Benzyl-Arg-Gly-NH2 was set up as pH 8.0, 15 degrees C in 40% MeOH for 10 h with the maximum yield of 73.6%. The optimum condition for the synthesis of CBZ-Gly-Asp-NH2 was pH 7.0, 15 degrees C in 50% MeOH for 10h with the maximum yield of 67.0%.  相似文献   

9.
α-Chymotrypsin (α-CT; EC 3.4.21.1) and papain (EC 3.4.22.2) were used as catalysts in preparative peptide synthesis in frozen aqueous systems. A special apparatus was constructed in order to enable shock freezing of large reaction volumes. Several hundred milligram of peptides including non-natural components were synthesized representing yields of 43% to 95%.  相似文献   

10.
    
Under ordinary conditions the equilibrium point in protease-catalyzed reactions is near to complete hydrolysis. Therefore, proteases are commonly known for their proteolytic rather than for their proteosynthetic activities. Nevertheless, the proteases have proved to be excellent catalysts in preparative peptide synthetic chemistry. A brief review is given of the historical development of protease-catalyzed peptide synthesis. The theoretical aspects of peptide bond formation are described and particular emphasis is given to techniques for favoring the synthesis of the desired peptide linkages. The applicability of these techniques is exemplified with selected syntheses and semisyntheses. The advantages as well as the problems associated with the enzymatic method are evaluated. A critical assessment is given of the present state of the art and the perspectives of the enzymatic approach to peptide synthetic chemistry.  相似文献   

11.
Under ordinary conditions the equilibrium point in protease-catalyzed reactions is near to complete hydrolysis. Therefore, proteases are commonly known for their proteolytic rather than for their proteosynthetic activities. Nevertheless, the proteases have proved to be excellent catalysts in preparative peptide synthetic chemistry. A brief review is given of the historical development of protease-catalyzed peptide synthesis. The theoretical aspects of peptide bond formation are described and particular emphasis is given to techniques for favoring the synthesis of the desired peptide linkages. The applicability of these techniques is exemplified with selected syntheses and semisyntheses. The advantages as well as the problems associated with the enzymatic method are evaluated. A critical assessment is given of the present state of the art and the perspectives of the enzymatic approach to peptide synthetic chemistry.  相似文献   

12.
The peptide bond formation of N-protected non-coded amino acids having different structures as acyl donor substrates that is catalyzed by thermoase in organic media was investigated. In these reactions, N-protected l--non-coded amino acids, including l-Orn, l-Cit, -aminobutyric acid (l--Abu) and phenylalanine homologues, were used as the acyl donors and phenylalanine derivatives were used as the acyl acceptors. This kind of enzymatic reactions cannot be carried out in an aqueous buffer due to the rigid specificity of proteases to coded amino acids in water. The results demonstrated that the substrate specificity of proteases could be broadened in organic solvents. In addition, the factors that influenced these protease-catalyzed reactions, including structures of the substrates, water content and the bases used, were systematically studied. Our work provided important evidence for broadening the application of protease in organic synthesis.  相似文献   

13.
M Mutter  H Mutter  R Uhmann  E Bayer 《Biopolymers》1976,15(5):917-927
The conformation of polyethylene glycol-bound peptides, synthesized by the liquid-phase method, was investigated. This marcromolecular C-terminal protecting group is transparent in the visible and the ultraviolet range to 190 nm and solubilizes peptides in many different solvents. The CD spectra of the polymer-bound myoglobin sequence 66–73 and of the biologically active undecapeptide “substance P” were measured in each step of the synthesis. In both examples the formation of a secondary structure during the growth of the peptide chain was found. In the hydrophobic octapeptide containing the myoglobin sequence 66–73, the influence of either the blocked or the free N-terminal amino group on the conformation was observed. The blocked octapeptide in trifluoroethanol showed a higher degree of α-helix contribution than in its free state. The conformation of the polyethylene glycol-bound nona- and decaalanine in trifluoroethanol and water was determined. The peptide with a free amino end group has β-conformation in trifluoroethanol as well as in water. The corresponding N-Boc-protected derivatives show helical structure. The amino end group has a decisive influence on the formation of β-structure. The method of CD investigation of polymer-bound peptide sequences during the peptide synthesis in solution enables one to determine the influence of protecting groups and the chain end of a peptide on its conformation. It is also possible to study the relationship between the secondary structure, the chain length, and the kinetic of the coupling reaction in different solvents. Since the crystallization method for the liquid-phase peptide synthesis allows one to synthesize peptides in very short time, a new method of studying peptide conformations is opened.  相似文献   

14.
Enzymatic synthesis of Leu- and Met-enkephalin   总被引:1,自引:0,他引:1  
The protease-catalyzed synthesis of Leu- and Met-enkephalin is reported. Each peptide bond of the endogeneous opiate-pentapeptides was formed either by papain or α-chymotrypsin catalysis. N-acyl amino acids and peptides or their ester derivatives served as substrates whereas amino acid and peptide phenylhydrazides were used as nucleophiles. The free pentapeptides exhibited naloxone-reversible opiate-like activity in guinea-pig ileum and mouse vas deferens assays. The present study suggests the usefulness of enzymic peptide synthesis which allows rapid preparation of homogeneous compounds with high optical purity.  相似文献   

15.
The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH(2) was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-alpha protection choice. Boc and Z were the most reactive N-alpha groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.  相似文献   

16.
To study the influence of charges on the partition of solutes in aqueous two-phase systems of polyethylene glycol and dextran, partition coefficients of dimethylaminoethyl-dextran, trimethylamino-dextran, and bis (alpha,omega)-amino-poly(ethylene glycol) were determined as a function of pH (range 2 to 12) and ionic strength. These polymers are derivatives of the phase forming components and carry ionizable groups that are charged or uncharged depending on the pH. Unexpectedly, the largest differences in the partition coefficients were found at high pH, where the modified polymers are uncharged. In addition, the partitioning of low-molecular-weight model compounds, ethylenediamine and iminodiacetic acid, as well as poly-L-lysine and poly(allylamine) was analyzed. A consistent pattern was observed in the partition of polyelectrolytes reflecting the influence of charge, but another property of aqueous phase systems unrelated to charge and changing with pH seems to be superimposed. (c) 1995 John Wiley & Sons, Inc.  相似文献   

17.
This study examined the solubility enhancement of 4 cox-2 inhibitors, celecoxib, rofecoxib, meloxicam, and nimesulide, using a series of pure solvents and solvent mixtures. Water, alcohols, glycols, glycerol, and polyethylene glycol 400 (PEG 400) were used as solvents and water-ethanol, glycerol-ethanol, and polyethylene glycol-ethanol were used as mixed-solvent systems. A pH-solubility profile of drugs was obtained in the pH range 7.0 to 10.9 using 0.05M glycine-sodium hydroxide buffer solutions. Lower alcohols, higher glycols, and PEG 400 were found to be good solvents for these drugs. The aqueous solubility of celecoxib, rofecoxib, and nimesulide could be enhanced significantly by using ethanol as the second solvent. Among the mixed-solvent systems, PEG 400-ethanol system had highest solubilization potential. In the case of meloxicam and nimesulide, solubility increased significantly with increase in pH value. Physico-chemical properties of the solvent such as polarity, intermolecular interactions, and the ability of the solvent to form a hydrogen bond with the drug molecules were found to be the major factors involved in the dissolution of drugs by pure solvents. The greater the difference in the polarity of the 2 solvents in a given mixed solvent, the greater was the solubilization power. However, in a given mixed-solvent system, the solubilization power could not be related to the polarity of the drugs. Significance of the solubility data in relation to the development of formulations has also been discussed in this study.  相似文献   

18.
The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in the site-specific amino acid moiety from the acyl residue to the ester-leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid-phase peptide synthesis with substrate mimetic-mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4-guanidinophenyl-, phenyl- and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N(alpha)-protected peptide esters as acyl donors in trypsin-, alpha-chymotrypsin- and V8 protease-catalyzed fragment condensations.  相似文献   

19.
Pore formation constitutes a key step in the mode of action of Bacillus thuringiensis delta-endotoxins and various activated Cry toxins have been shown to form ionic channels in receptor-free planar lipid bilayers at high concentrations. Multiple conductance levels have been observed with several toxins, suggesting that the channels result from the multimeric assembly of a variable number of toxin molecules. To test this possibility, the size of the channels formed by Cry1C was estimated with the non-electrolyte exclusion technique and polyethylene glycols of various molecular weights. In symmetrical 300 mM KCl solutions, Cry1C induced channel activity with 15 distinct conductance levels ranging from 21 to 246 pS and distributed in two main conductance populations. Both the smallest and largest conductance levels and the mean conductance values of both populations were systematically reduced in the presence of polyethylene glycols with hydrated radii of up to 1.05 nm, indicating that these solutes can penetrate the pores formed by the toxin. Larger polyethylene glycols had little effect on the conductance levels, indicating that they were excluded from the pores. Our results indicate that Cry1C forms clusters composed of a variable number of channels having a similar pore radius of between 1.0 and 1.3 nm and gating synchronously.  相似文献   

20.
The physicochemical mechanism of protease-catalyzed peptide synthesis in heterogenous etuectic mixtures of substrates has been examined by a combination of microscopic techniques. Using a number of model reactions of dipeptide amide synthesis, it was determined that liquid phase catalysis was mostly, if not exclusively, responsible for the observed conversion of substrates. Furthermore, the formation of liquid or semiliquid eutectics was an important requirement for the occurrence of those reactions where both substrates were solids in the pure state. The addition of small quantities of hydrophilic solvents (adjuvants) often resulted in significat improvements in the rates and yields of the reactions. This was due to the ability of these adjuvants to promote the formation of eutectics, thereby increasing the proportion, as well as affecting the composition the properties, as well as affecting the composition and properties of the liquid phase. (c) 1994 John Wiley & Sons, Inc.  相似文献   

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