共查询到20条相似文献,搜索用时 31 毫秒
1.
Bacheva A. V. Baibak O. V. Belyaeva A. V. Lysogorskaya E. N. Oksenoit E. S. Lozinskii V. I. Filippova I. Yu. 《Russian Journal of Bioorganic Chemistry》2003,29(5):502-508
The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content >80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, Lys, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70–98% in DMF–MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids. 相似文献
2.
alpha-Chymotrypsin (CT), subtilisin BPN' (STB), and subtilisin Carlsberg (STC) were immobilized by adsorption to porous chitosan beads (Chitopearl, CP). The immobilized enzymes showed higher catalytic activities than free enzymes for amino acid esterification in many hydrophilic organic solvents except for methanol and DMF. In ethanol, the initial rate of the esterification increased with water content, whereas in ethyl acetate, the maximum rate was obtained at 2%-3% water. CP-immobilized CT also catalysed transesterification of Ac-Tyr-OMe in ethanol and peptide synthesis in acetonitrile from Ac-Tyr-OH or its ethyl ester and amino acid amides. The immobilized enzymes are highly stable in organic solutions, and can easily be separated from the reaction solutions. Repeated esterifications of Ac-Tyr-OH in acetonitrile by a CP-immobilized CT gave almost constant yields of the ester for more than 3 weeks. 相似文献
3.
Subtilisin Carlsberg, an alkaline protease from Bacillus licheniformis, was modified with polyoxyethylene (PEG) or aerosol-OT (AOT), and the solubility, conformation, and catalytic activity of the modified subtilisins in some organic media were compared under the same conditions. The solubility of modified subtilisins depended on the solubility of the modifier. On the other hand, the conformational changes depended on the solubility, rather than the property, of the modifier. When the modified subtilisin was dissolved in water-miscible polar solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran, significant conformational changes occurred. When modified subtilisin was dissolved in water-immiscible organic solvents, such as isooctane and benzene, the solvent did not induce significant conformational changes. The catalytic activity in the transesterification reaction of the N-acetyl-L-phenylalanine ethylester of the modified subtilisin in organic solvents was higher than that of native subtilisin. The high activity of modified subtilisin was thought to be due to a homogeneous reaction by the dissolved enzymes. 相似文献
4.
We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp. 相似文献
5.
We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp. 相似文献
6.
The catalytic feature of serine proteases for synthetic reactions in hydrophilic organic solvents and effects of immobilization by complexation with polysaccharides are described. Free alpha-chymotrypsin and subtilisin Carlsberg catalyze esterification, transesterification, and peptide synthesis in hydro-organic cosolvents with less than 10% water. Subtilisin BPN' is catalytically less active. The medium effects on the reaction kinetics and product yield were investigated in terms of the nature of solvent and water content in the reaction systems. The substrate- and stereo-specificities of the enzymes suggest that the enzymes maintain their native conformations in these low-water organic solvents. The catalytic activities of the proteases markedly increase by immobilization or complexation with polysaccharides, such as chitin or chitosan. The results of the rate measurements suggest that the primary role of the support materials is the activation of the enzymes and the increase in substrate concentration at reaction sites. 相似文献
7.
Bacheva AB Isakov MS Lysogorskaya EN Macquarrie DJ Filippova IIu 《Bioorganicheskaia khimiia》2008,34(3):371-375
New biocatalysts, preparations of subtilisin Carlsberg immobilized on chitosan (a deacetylated derivative of chitin), were obtained. The enzyme content, hydrolytic activity, and ability to catalyze peptide bond formation in organic solvents were characterized for these preparations. The influence of the form and composition of the biocomplex (content of the enzyme and glutaraldehyde, the cross-linking agent) and buffer pH on the biocata-lytic properties of the immobilized enzyme was studied in the reactions of peptide bond hydrolysis. The synthase activity of the preparations was investigated in the reaction of synthesis of Z-Ala-Ala-Leu-Phe-pNA in a 6 : 4 DMF-acetonitrile mixture in dependence on the reaction time. The yield of this product was 100% after only 40 min. 相似文献
8.
A. V. Bacheva M. S. Isakov E. N. Lysogorskaya D. J. Macquarrie I. Yu. Philippova 《Russian Journal of Bioorganic Chemistry》2008,34(3):334-338
New biocatalysts, preparations of subtilisin Carlsberg immobilized on chitosan (a deacetylated derivative of chitin), were obtained. The enzyme content, hydrolytic activity, and ability to catalyze peptide bond formation in organic solvents were characterized for these preparations. The influence of the form and composition of the biocomplosite (content of the enzyme and glutaraldehyde, the cross-linking agent) and buffer pH on the biocatalytic properties of the immobilized enzyme was studied in the reactions of peptide bond hydrolysis. The synthase activity of the preparations was investigated in the reaction of synthesis of Z-Ala-Ala-Leu-Phe-pNA in a 6:4 DMF-acetonitrile mixture in dependence on the reaction time. The yield of this product was 100% after only 40 min. 相似文献
9.
Anikina OM Semashko TA Oksenoĭt ES Lysogorskaia EN Filippova IIu 《Bioorganicheskaia khimiia》2006,32(2):130-136
The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z- ALA-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P1 and P'1 (Glu, Arg, and Asp) containing unprotected side ionogenic groups. 相似文献
10.
The serine proteases alpha-chymotrypsin, trypsin, and subtilisin Carlsberg were immobilized in a sol-gel matrix and the effects on the enzyme activity in organic media are evaluated. The percentage of immobilized enzyme is 90% in the case of alpha-chymotrypsin and the resulting specific enzyme activity in the transesterification of N-acetyl-L-phenylalanine ethyl ester with 1-propanol in cyclohexane is 43 times higher than that of a nonimmobilized lyophilized alpha-chymotrypsin. The activities of trypsin and subtilisin Carlsberg are enhanced with 437 and 31 times, respectively. The effect of immobilization on the enzyme activity is highest in hydrophobic solvents. 相似文献
11.
Summary Subtilisin Carlsberg and subtilisin BPN' (nagarse) catalyze peptide bond formation from aromatic amino acid esters and glycinamide in hydrophilic organic solvents. The activities of subtilisin and product compositions are different in several organic solvents; reactions in acetonitrile, tetrahydrofuran, and propylene carbonate gave the peptide in excellent yields, while in N,N-dimethylformamide and methanol the enzyme activity was largely retarded. The yield of the peptide is also dependent on water content in the reaction solutions. Optimum water contents are in the range from 3 to 7 %. The reaction is strongly specific for glycinamide as an amine component, and amides of alanine, valine, and leucine gave the corresponding peptides in poor yields. 相似文献
12.
Anna V. Bacheva Anastasiya V. Belyaeva Elena N. Lysogorskaya Elena S. Oksenoit Vladimir I. Lozinsky Irina Yu. Filippova 《Journal of Molecular Catalysis .B, Enzymatic》2005,32(5-6):253-260
Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM. 相似文献
13.
A series of N-protected peptide alcohols were synthesized using amino alcohols with unprotected hydroxy groups as amino components by the catalysis of subtilisin or alpha-chymotrypsin in organic solvents. N-protected aromatic amino acid esters were more suitable as acyl donors for subtilisin. The influences of different N-protecting groups, organic solvents, and content of water on synthesis of N-protected peptide alcohols were systematically studied. 相似文献
14.
Trypsin was covalently immobilized on porous glass in the presence and absence of a specific substrate and reacted in various organic solvents of different dielectric constants. Optimum solvent concentration, pH profile, Km(app), Vmax(app), productivity versus temperature, activity, and reaction rates were determined. Reaction rates of six lysyl dipeptides were compared. Crystalline trypsin was dansylated for studies by nanosecond fluorescence techniques to determine the effects of introducing high concentrations of organic solvents on the molecule. The results indicated that greater reaction rates were observed with dipeptides having more acidic carboxyl terminal groups. The data also indicated that greater reaction rates were observed in higher concentrations of solvents of lower dielectric constants. Nanosecond fluorescence spectroscopy of trypsin in high concentrations of a low dielectric constant solvent indicated major dehydration even though maximal enzyme activity was achieved under these conditions. 相似文献
15.
Simple co-lyophilization of serine protease subtilisin Carlsberg with [12]-crown ether-4 (12-crown-4) or methyl-beta-cyclodextrin (MbetaCD) drastically increases its catalytic activity in organic solvents. We investigated whether the improved activity would cause substrate diffusional limitations. To experimentally assess the issue, the enzyme was inactivated with PMSF. Different amounts of active and inactive subtilisin were codissolved in 10 mM phosphate buffer (pH 7.8) followed by lyophilization with or without 12-crown-4 or MbetaCD. Initial rates for the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester and 1-propanol in anhydrous THF were plotted vs. the amount of active enzyme present in the formulations. For all three enzyme formulations a linear relationship was observed and the results clearly show that activation of subtilisin Carlsberg by crown ethers and MbetaCD did not cause diffusional limitations. This was somewhat surprising because theoretical models predicted such diffusional limitations for the activated formulations. However, investigation of the protein powder particles obtained after co-lyophilization with 12-crown-4 and MbetaCD revealed a drastically reduced particle size for these formulations when suspended in THF. The particle micronization afforded by the excipients prevented substrate diffusional limitations, a factor that should be taken into account when designing improved enzyme formulations for synthetic applications in organic solvents. 相似文献
16.
O. M. Anikina T. A. Semashko E. S. Oksenoit E. N. Lysogorskaya I. Yu. Filippova 《Russian Journal of Bioorganic Chemistry》2006,32(2):116-121
The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z-Ala-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P 1 and P 1 ′ (Glu, Arg, and Asp) containing unprotected side ionogenic groups. 相似文献
17.
Steady-state kinetic analysis has been used to confirm the catalytic mechanism of lyophilized subtilisin suspended in a variety of organic solvents. Specifically, this article demonstrates that partial reactions can occur between subtilisin and ester substrates in organic solvents. Partitioning of common intermediates between competing acceptors at a constant ratio of products has also been described. The decomposition of a common intermediate formed from different substrates at the same rate is also further evidence of an acyl-enzyme mechanism for subtilisin suspended in anhydrous solvents. Partitioning of a common intermediate to give two products at a constant total rate, and saturation kinetics at varying substrate concentrations, complete a kinetic investigation of the enzyme mechanism. All the data generated support the formation of a stable acyl enzyme during the transesterification reaction catalzyed by subtilisin in the solvents used. 相似文献
18.
Anne Ferjancic Antoine Puigserver Hubert Gaertner 《Applied microbiology and biotechnology》1990,32(6):651-657
Summary Subtilisin from Bacillus subtilis was modified with polyethylene glycol (PEG), or adsorbed either on celite or porous glass, or directly used as a suspended powder to catalyse peptide synthesis and transesterification reactions in organic solvents. The rather low yield of peptide synthesis probably resulted from the enzyme tendency to catalyse hydrolysis and transesterification side reactions. The kinetics of transesterification catalysed by PEG-subtilisin was consistent with a ping-pong mechanism modified by a hydrolytic branch. Initial rates of transesterification were found to be dependent on alcohol and organic base concentrations in the reaction mixture. The high affinity of benzyloxycarbonyl-l-serine-methyl ester for the enzyme indicated that a change in substrate specificity of subtilisin occurred in organic phase. The 50-fold increase in the rate of synthesis of benzyloxycarbonyl-l-serine-l-phenylalanine amide which was observed when PEG-subtilisin was used instead of immobilized or powdered enzyme, suggested that a higher flexibility of the polypeptide chain modified by the covalent attachment of a number of soluble PEG moieties occurred in organic solvents. This also resulted in a lower stability of PEG-subtilisin at high temperature.Offprint requests to: A. Puigserver 相似文献
19.
The substrate specificities of alpha-chymotrypsin and subtilisins for peptide synthesis in hydrophilic organic solvents were investigated. Chymotrypsin exhibited high specificity to aromatic amino acids as acyl donors, while subtilisin Carlsberg and subtilisin BPN' were specific to aromatic and neutral aliphatic amino acids, in accordance with the S1 specificities of the enzymes for peptide hydrolysis in aqueous solutions. On the contrary, chymotrypsin exhibited higher specificities to hydrophilic amino acid amides as acyl acceptors (nucleophiles) for peptide synthesis with N-acetyl-L-tyrosine ethyl ester, in contrast to the S1' specificity for peptide hydrolysis and peptide synthesis in aqueous solutions. Furthermore, nucleophile specificity changed with the change in water-organic solvent composition; the increase in water content led to increase in relative reactivity of leucinamide to that of alaninamide. It was also found that protection of the carboxyl group of alanine by amidation is much preferable to protection by esterification in terms of reactivity as nucleophiles. 相似文献
20.
The protease-catalyzed transesterifications between N-trifluoroacetyl-DL-phenylalanine 2,2,2-trifluoroethyl ester and 1-propanol were studied in a variety of anhydrous organic solvents at 30 degrees C. The protease preparations lyophilized from phosphate buffer solutions (pH 8.0) were used as catalysts. The organic solvent affected both rate of reaction and enantioselectivity differently. Proteases such as Aspergillus oryzae protease, subtilisin Carlsberg, and subtilisin BPN' always preferred the L-enantiomer in both hydrophilic and hydrophobic solvents, indicating no inversion of the L-specificity in hydrophobic solvents such as toluene. However, enantioselectivity was rather poor, with E (enantiomeric ratio) values not exceeding even one order of magnitude except for acetonitrile. There was a weak inverse correlation between E values of subtilisin Carlsberg and solvent hydrophobicity (logP). Acetonitrile was a preferable solvent in terms of both rate of reaction and enantioselectivity (E= 15 to 25) for processing L-amino acid derivatives in organic media. Organic solvents generally have potential advantages of processing D-amino acid derivatives. (c) 1997 John Wiley & Sons, Inc. 相似文献