- Study the effect of trypsin enzyme activity on the screening of applying frontal affinity chromatography
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Frontal affinity chromatography (FAC) combined with enzyme has been widely used for drug screening. In this paper, the effect of target enzyme activity on screening of bioactive compounds was studied through applying FAC. Trypsin with different degree of inactivation were prepared as target enzyme by thermal denaturation. Their primary structure was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and use Fourier transform infrared (FTIR) and ultraviolet-visible (UV–vis) spectroscopy to detect group structure. Ultimately, it was found that the main structure of enzyme with decreased activity remained unchanged. The oxymatrine and matrine which can interact with trypsin were selected to study their binding to trypsin with different activities in FAC. The results showed that oxymatrine and matrine had a significant difference in the breakthrough volume among seven kinds of columns prepared by trypsins with different activities, at the different concentration. It indicated that trypsins with different activities in FAC could combine with oxymatrine and matrine. The binding constant (Kd) variation between oxymatrine, matrine and trypsin with different activities are 5.520 ± 0.038 and 3.577 ± 0.071, within error range, which indicated that the activity of target enzyme with primary unchanged structure has no effect on screening of bioactive components by FAC.
- Qian, JunQing,Zhao, ChangYan,Tong, Jun,Jiang, ShengLan,Zhang,Lu, ShiYong,Guo, Hui
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- Synthesis of papain-polyacrylamide hydrogel microspheres and their catalytic application
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Although immobilized enzyme technologies effectively improve the reusability and stability of an enzyme, the introduction of covalent bonds or/and charge interaction causes the change for the secondary structure of an enzyme, thereby reducing the catalytic activity. In this work, a mild and efficient strategy for enzyme immobilization is proposed. A size-controlled hydrogel microsphere was designed and synthesized as an immobilized enzyme carrier. Papain was immobilized on the surface and pores of hydrogel microspheres by hydrogen bonding interactions, which have no adverse effect on the secondary structure of an enzyme. The activity of the papain-polyacrylamide hydrogel microsphere was 106.41% of that of free papain. In addition, the immobilized papain maintained 59.60% of free papain activity after 10 cycles. The results of practical catalytic application proved that the obtained papain-polyacrylamide hydrogel microspheres were very suitable for catalytic hydrolysis of allergen protein in cow's milk without introducing any other hetero protein.
- Fei, Xu,Li, Yao,Ma, Yuan,Tian, Jing,Wang, Yi,Xu, Longquan
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- Biocatalytic synthesis, antimicrobial properties and toxicity studies of arginine derivative surfactants
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Abstract Two novel arginine-based cationic surfactants were synthesized using as biocatalyst papain, an endopeptidase from Carica papaya latex, adsorbed onto polyamide. The classical substrate N α-benzoyl-arginine ethyl ester hydrochloride for the determination of cysteine and serine proteases activity was used as the arginine donor, whereas decyl- and dodecylamine were used as nucleophiles for the condensation reaction. Yields higher than 90 and 80 % were achieved for the synthesis of N α-benzoyl-arginine decyl amide (Bz-Arg-NHC10) and N α-benzoyl-arginine dodecyl amide (Bz-Arg-NHC12), respectively. The purification process was developed in order to make it more sustainable, by using water and ethanol as the main separation solvents in a single cationic exchange chromatographic separation step. Bz-Arg-NHC10 and Bz-Arg-NHC12 proved antimicrobial activity against both Gram-positive and Gram-negative bacteria, revealing their potential use as effective disinfectants as they reduced 99 % the initial bacterial population after only 1 h of contact. The cytotoxic effect towards different cell types of both arginine derivatives was also measured. Bz-Arg-NHCn demonstrated lower haemolytic activity and were less eye-irritating than the commercial cationic surfactant cetrimide. A similar trend could also be observed when cytotoxicity was tested on hepatocytes and fibroblast cell lines: both arginine derivatives were less toxic than cetrimide. All these properties would make the two novel arginine compounds a promising alternative to commercial cationic surfactants, especially for their use as additives in topical formulations.
- Fait, M. Elisa,Garrote, Graciela L.,Clapés, Pere,Tanco, Sebastian,Lorenzo, Julia,Morcelle, Susana R.
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- Enzyme kinetics in crowded solutions from isothermal titration calorimetry
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Isothermal titration calorimetry (ITC) is a universal technique that directly measures the heat absorbed or released in a process. ITC is typically used to determine thermodynamic parameters of association of molecules without the need to label them. However, ITC is still rarely applied to study chemical reactions catalyzed by enzymes. In addition, these few studies of enzyme kinetic measurements that have been performed were in diluted solutions. Yet, to estimate realistic kinetic parameters, we have to account for the fact that enzymatic reactions in cells occur in a crowded environment because cells contain 200–400 g/L of macromolecular crowders such as proteins, ribosomes and lipids. Thus we expanded the ITC application for solutions mimicking the cellular environment by adding various macromolecular crowders. We investigated how these crowders affect the kinetics of trypsin-catalyzed reactions and determined the Michaelis-Menten parameters for hydrolysis of two trypsin substrates: Nα-benzoyl-L-arginine ethyl ester (BAEE) and Nα-benzoyl-DL-arginine β-naphthylamide (BANA). Since ITC enables investigations of complex and turbid solutions with label-free reagents, it seems a perfect technique for kinetic analyses in crowded solutions. ITC also offers the opportunity to control enzyme-crowder and substrate-crowder interactions.
- Maximova, Ksenia,Wojtczak, Jakub,Trylska, Joanna
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- Time-resolved dynamic nuclear polarization enhanced NMR spectroscopy
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(Chemical Presented) The sensitive touch: Sensitivity constraints in NMR spectroscopy typically call for long measurement times. Hyperpolarization can enhance the time resolution of NMR spectroscopy by removing the need for signal averaging. Reactions such as enzyme catalysis can be followed in real time by hyperpolarized NMR spectroscopy through reduction in the intensity of the substrate resonance as well as the appearance of product resonances (see picture).
- Bowen, Sean,Hilty, Christian
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- Comparative Studies on the Interaction of Spermidine with Bovine Trypsin by Multispectroscopic and Docking Methods
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The effect of spermidine on the kinetics, conformation, and dynamics of native trypsin was studied by steady-state thermal stability, intrinsic fluorescence, circular dichroism (CD), ultraviolet-visible (UV-vis) spectroscopy, and kinetic techniques, as well as molecular docking, at the temperatures of 298 and 308 K. The Stern-Volmer quenching constants (Ksv) for the trypsin-spermidine complex were obtained at two temperatures, revealing that spermidine quenched the intensity of trypsin through the static mode of the quenching mechanism. The corresponding thermodynamic parameters, Gibbs free-energy, enthalpy, and entropy changes, showed that the binding process was spontaneous. These values and the molecular docking technique revealed that the hydrogen bonding and van der Waals forces played a major role in stabilizing the complex. CD, absorption, and fluorescence results also indicated that spermidine binding had a partial effect on trypsin structure. Spermidine could also influence the activity of trypsin. Upon spermidine binding, the Vmax value of the enzyme was increased and the kcat/Km values were enhanced slightly. The Tm of the trypsin-spermidine complex was enhanced probably due to the higher H-bond formation and lower surface hydrophobicity after spermidine modification, as confirmed by UV-vis spectroscopy and fluorescence spectra. UV absorption and CD studies also indicated that the binding of spermidine to trypsin had induced microenvironmental changes around the enzyme, leading to changes in its secondary structure.
- Momeni, Lida,Shareghi, Behzad,Saboury, Ali Akbar,Farhadian, Sadegh
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- Synthetic approaches to a challenging and unusual structure—an amino-pyrrolidine guanine core
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The synthesis of an unreported 2-aminopyrrolidine-1-carboxamidine unit is here described for the first time. This unusual and promising structure was attained through the oxidative decarboxylation of amino acids using the pair of reagents, silver(I)/peroxydisulfate (Ag(I)/S2O82?) followed by intermolecular (in the case of L-proline derivative) and intramolecular trapping (in the case of acyl L-arginine) by N-nucleophiles. The L-proline approach has a broader scope for the synthesis of 2-aminopyrrolidine-1-carboxamidine derivatives, whereas the intramolecular cyclization afforded by the L-acylarginines, when applied, results in higher yields. The former allowed the first synthesis of cernumidine, a natural alkaloid isolated in 2011 from Solanum cernuum Vell, as its racemic form.
- Rippel, Rafael,Pinheiro, Luís,Lopes, Mónica,Louren?o, Ana,Ferreira, Luísa M.,Branco, Paula S.
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- Pyrazine-derived disulfide-reducing agent for chemical biology
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For fifty years, dithiothreitol (DTT) has been the preferred reagent for the reduction of disulfide bonds in proteins and other biomolecules. Herein we report on the synthesis and characterization of 2,3-bis(mercaptomethyl)pyrazine (BMMP), a readily accessible disulfide-reducing agent with reactivity under biological conditions that is markedly superior to DTT and other known reagents. This journal is the Partner Organisations 2014.
- Lukesh, John C.,Wallin, Kelly K.,Raines, Ronald T.
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supporting information
p. 9591 - 9594
(2014/08/18)
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- Nanostructured layered double hydroxide aerogels with enhanced adsorption properties
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Aerogels of layered double hydroxides were prepared by a simple and eco-friendly method involving a quick coprecipitation followed by supercritical CO2 drying. Such aerogels display high surface areas and enhanced adsorption behavior.
- Touati, Souad,Mansouri, Hela,Bengueddach, Abdelkader,De Roy, Andre,Forano, Claude,Prevot, Vanessa
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supporting information; scheme or table
p. 7197 - 7199
(2012/08/13)
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- Encapsulation of enzyme in large mesoporous material with small mesoporous windows
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Trypsin has been encapsulated in the mesopores of a hierarchical mesoporous silica material synthesized via Cu(i) catalyzed azide-alkyne click reaction between azide functionalized large spherical SBA-15 particles and alkyne functionalized mesoporous sili
- Malvi, Bharmana,Gupta, Sayam Sen
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supporting information; experimental part
p. 7853 - 7855
(2012/09/05)
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- Utilization of the IC-calorimeter for study of enzymatic reaction
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In this article, the monitoring of an enzymatic reaction by means of a miniaturized batch type IC-calorimeter was performed. The aim of this work was focused on an investigation of enthalpy and rate of enzymatic reaction of trypsin with Nα-benzoyl-l-arginine-p-nitroanilide hydrochloride (BApNA). Both the parameters were determined for reactions in different buffers and for varying concentration of enzyme at 37 °C. The rate of reaction decreased with the increasing concentration of enzyme caused by trypsin autolysis.
- Podzemna, V.,Slovakova, M.,Kourkova, L.,Svoboda, L.
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experimental part
p. 715 - 719
(2011/01/09)
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- PEPTIDYL ARGININE DEIMINASE TYPE IV INHIBITOR
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A compound represented by the general formula (I) or a salt thereof is provided: wherein R1, R2 and R3 each independently represent a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, provided that at least one of R1, R2 and R3 does not represent a hydrogen atom; R4 represents an amino group which has a substituent; and R5 represents a carboxyl group which may have a substituent. Also provided is a peptidylarginine deiminase 4 inhibitor. The inhibitor can be used for the prevention and/or treatment of diseases associated with peptidylarginine deiminases (e.g., rheumatoid arthritis and multiple sclerosis).
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Page/Page column 20-21
(2008/06/13)
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- Effect of freezing on the enzymatic coupling of specific amino acid-containing peptide fragments
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The effect of freezing on the enzymatic coupling of highly specific amino acid-containing peptide fragments was investigated using trypsin, α-chymotrypsin, and Bacillus licheniformis Glu-specific endopeptidase as biocatalysts. Comparison with reactions at normal temperature indicates that freezing efficiently represses the cleavage of specific peptide bonds independent of their individual localisation and specificity achieving irreversible and efficient peptide bond formation without proteolytic side reactions. Copyright (C) 2000 Elsevier Science Ltd.
- Wehofsky, Nicole,Haensler, Marion,Kirbach, Sebastian W.,Wissmann, Johannes-Dieter,Bordusa, Frank
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p. 2421 - 2428
(2007/10/03)
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- Investigations on the Enzyme Specificity of Clostripain: A New Efficient Biocatalyst for the Synthesis of Peptide Isosteres
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To explore the ability of the cysteine protease clostripain as a biocatalyst for the synthesis of peptide isosteres, the S′-subsite specificity of this enzyme toward unnatural substrates was investigated. First, the function of clostripain for acylating aliphatic noncyclic and cyclic amines varying in chain length and ring size was analyzed using a standard acyl donor. Additionally, this series was expanded by use of aromatic amines, amino alcohols, derivatives of non-α-amino carboxylic acids, and symmetric and asymmetric diamines, respectively. The results obtained give a detailed picture of the unique reactivity of clostripain toward synthetic substrates, allowing insights into the basic enzyme-substrate interactions. Furthermore, the data provide a guideline for the use of clostripain as a biocatalyst for synthesis of peptide isosteres. The study was completed by the utilization of a model substrate mimetic enabling clostripain to react with noncoded and non-amino acid-derived amines as well as nonspecific acyl moieties. The results of this study indicate that this approach may extend the application range of clostripain as a biocatalyst outside of peptide synthesis.
- Guenther, Robert,Stein, Anja,Bordusa, Frank
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p. 1672 - 1679
(2007/10/03)
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- Isolation and some properties of a serine protease from the fruits of Cudrania cochinchinensis (Lour.) Kudo et Masam.
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An endopeptidase (Cudrania protease) with a molecular mass of 76 kDa has been purified from the fruits of Cudrania cochinchinensis (Lour.) Kudo et Masam. The enzyme was stable between pH 6 and 10 at 30 degrees C for 60 min. The enzyme activity was inhibit
- Uchikoba,Arima,Shimada,Yonezawa,Kaneda
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p. 623 - 624
(2007/10/03)
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- Nucleophile specificity in trypsin-catalyzed acyl transfer using non-specific acyl donors
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The S'-subsite specificity of trypsin has been studied by comparing the non-specific acyl donors Mal-Phe-OCam (Mal = maleyl, OCam = carboxamidomethyl ester), Mal-Gly-Phe-OCam and Mal-Phe-OMe (OMe = methyl ester) with the specific substrate Bz-Arg-OEt (Bz = benzoyl, OEt = ethyl ester). Various nucleophilic amino components were chosen to determine S'1-P'1 interactions depending on the acyl donor used. The data obtained underline that there are no significant differences in the nucleophile specificity for specific and non-specific acyl donors, but with the latter a slightly better acceptance of the nucleophiles was found. Independent of the acyl donor used, a preference for methionine and arginine in the S'1-subsite of trypsin could be established. The results provide evidence that the binding site of the non-specific substrates in the active site of trypsin is not different to that of Bz-Arg-OEt.
- Salchert,Ullmann,Jakubke
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p. 710 - 714
(2007/10/03)
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- Low molecular weight proteins as carriers for renal drug targeting. Preparation of drug-protein conjugates and drug-spacer derivatives and their catabolism in renal cortex homogenates and lysosomal lysates
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Low molecular weight proteins (LMWPs) are known to be reabsorbed and catabolized primarily by the proximal tubular cells of the kidneys. As such, LMWPs might serve as drug carriers that release drugs site-specifically in the kidney. We tested this concept in vitro by coupling different drugs to the LMWP lysozyme both directly (amide bond) and via different spacers: oligopeptides (amide bond), (poly-)α-hydroxy acids (ester bond), and a pH sensitive cis-aconityl spacer (amide bond). The capability of the kidney to release the parent drug from such drug-spacer derivatives and drug-LMWP conjugates by enzymatic or chemical hydrolysis of the bond was tested by incubation experiments in renal cortex homogenates and lysosomal lysates. Directly coupled conjugates of terminal carboxyl group containing drugs and lysozyme were catabolized to single amino acids, but did not result in release of the parent drug. The amide bond between the drug and the final amino acid (lysine) appeared to be stable in the incubation milieu. Different oligopeptide spacers coupled to the drugs showed similar results: the oligopeptide itself was cleaved but the amide bond between the drug and different single amino acids remained untouched. Only amide bonds of derivatives of carboxylic drugs with peptide structures themselves were cleaved. Some of the directly coupled conjugates of terminal amino drugs and oligopeptides showed clear release of the parent drug whereas others were stable. Terminal amino drugs were rapidly released from an acid-sensitive cis-aconityl spacer. Terminal carboxyl group containing drugs were enzymatically released from their glycolic and lactic ester spacers at different rates. These kinds of drugs were also released as parent drug from LMWP conjugates with ester spacers like L-lactic acid. Increasing spacer length by intercalating a tetra(L-lactic acid) molecule between the drug and the protein further increased the extent and rate of drug release, indicating increased accessability of the bond to the enzymes. Terminal amino group containing drugs were rapidly generated as parent drug from LMWP conjugates using an acid-sensitive spacer. In addition the conjugates were found to be adequately stable in plasma, considering their rapid clearance from the bloodstream. It is concluded that LMWPs may indeed be of use as carriers for specific renal delivery of drugs, since renal cortex homogenates and lysosomal lysates are able to catabolize the protein and generate the parent drug from drug-LMWP conjugates bearing suitable spacers. The option of enzymatic release is limited by the narrow specificity of the lysosomal enzymes. This has profound implications for the synthesis of suitable conjugates, since the nature of the drug itself, the type of bond and also spacer length largely determine whether release of the parent drug will occur. Tailor-made spacers containing the correct mode of attachment and the right spacer length are required for this option. Chemical hydrolysis using acid-sensitive linkers, is suggested as a viable alternative approach.
- Franssen,Koiter,Kuipers,Bruins,Moolenaar,De Zeeuw,Kruizinga,Kellogg,Meijer
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p. 1246 - 1259
(2007/10/02)
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- Enzymes in organic synthesis: Use of subtilisin and a highly stable mutant derived from multiple site-specific mutations
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A subtilisin mutant (subtilisin 8350) derived from subtilisin BPN' via six-specific mutations (Met50Phe, Gly169Ala, Asn76Asp, Gln206Cys, Tyr217Lys, and Asn218Ser) was found to be 100 times more stable than the wild-type enzyme in aqueous solution at room temperature and 50 times more stable than the wild type in anhydrous dimethylformamide. Kinetic studies using ester, thio ester, and amide substrates, and the transition-state analogue inhibitor Boc-Ala-Val-Phe-CF3, indicate the both the wild-type and the mutant enzymes have very similar specificities and catalytic properties. The inhibition constant (K(i)) = 5.0 μM) for the wild-type enzyme is approximately 5 times that of the mutant enzyme (K(i)) = 1.1 μM), suggesting that the mutant enzyme binds the reaction transition state more strongly than the wild-type enzyme. This result is consistent with the observed rate constants for the corresponding ester and amide substrates; i.e. the k(cat)/k(m) values for the mutant are larger than those for hhe wild-type enzyme. Application of the mutant enzyme and the wild-type enzyme to organic synthesis has been demonstrated in the regioselective acylation of nucleosides in anhydrous dimethylformamide (with 65-100% regioselectivity at the 5'-position), in the enantioselective hydrolysis of N-protected and unprotected common and uncommon amino acid esters in water (with 85-98% enantioselectivity for the L-isomer), and in the synthesis of di- and oligopeptides via aminolysis of N-protected amino acid and peptide esters. The enzymatic peptide synthesis was carried out under high concentrations of DMF (~50%) to improve substrate solubility and to minimize enzymatic peptide cleavage. Low enantioselectivity was observed in the enzymatic transformation of non-amino acid alcohols and acids.
- Wong,Chen,Hennen,Bibbs,Wang,L iu,Pantoliano,Whitlow,Bryan
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p. 945 - 953
(2007/10/02)
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- Acid-Sensitive Latent Inhibitors for Proteolytic Enzymes: Synthesis and Characterization
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The reaction between peptide aldehydes and acylhydrazones affords derivatives that represent potential prodrugs for selective inhibition of lysosomal enzymes.BzPheal=Ala, obtained from the reaction between N-benzoyl-L-phenylalaninal and N-acetyl-L-alanine hydrazide, has been most carefully studied.When BzPheal=Ala is introduced into ongoing reactions catalyzed by α-chymotrypsin or papain, the rate of these reactions diminishes more rapidly with time than do those of controls lacking BzPheal=Ala.Furthermore, the disparity between run and control is much greater at pH 5 than at pH 7.The extent of inhibition (defined as explained in the text) at pH 5 can exceed that at pH 7 by 25-40-fold.The data are quantitatively explained by a reaction scheme that recognizes three important properties of BzPheal=Ala: (1) It undergoes hydrolysis at pH 5-7 to regenerate N-benzoyl-L-phenylalaninal; (2) the aldehyde thus liberated is a far more potent inhibitor for serine or cysteine proteases than is BzPheal=Ala; and (3) the rate constant for hydrolysis of BzPheal=Ala at pH 5 greatly exceeds that at pH 7.
- Silver, Marc S.,Haskell, John H.
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p. 1253 - 1259
(2007/10/02)
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- On the Use of Carboxamidomethyl Esters in the Protease-Catalyzed Peptide Synthesis
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Carboxyamidomethyl esters (CAM esters) of Z-and Boc-protected alanine and phenylalanine were prepared in order to investigate their usefulness as substrates for α-chymotrypsin- and papain-catalyzed hydrolysis and peptide synthesis reactions.The easy removal of the CAM-C-protecting group under mild conditions and dependent on the enzyme specificity was demonstrated.Examples are given for the protease-catalyzed synthesis of various peptide derivatives using CAM esters as C- and N-components in aqueous-organic media.Comparatively short reaction times were observed. - Key words: Carboxyamidomethyl ester as C-protecting group; Enzymatic deprotection; Peptide synthesis; α-Chymotrypsin- and Papain-catalyzed peptide bond formation
- Kuhl, Peter,Zacharias, Ute,Burckhardt, Helmut,Jakubke, Hans-Dieter
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p. 1195 - 1204
(2007/10/02)
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- Peptide-bond Formation, Chemoselective Acylation of Amino Acids, and Crosslinking Reaction between Amino Acids Utilizing a Functional Five-membered Heterocycle, 1,3-Thiazolidine-2-thione
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The monitored aminolysis of 3-acyl-1,3-thiazolidine-2-thiones has been extended to the peptide-bond formation, the chemoselective acylation of amino acids having multifunctional groups, and the crosslinking reaction between amino acids.
- Nagao, Yoshimitsu,Miyasaka, Tadayo,Seno, Kaoru,Fujita, Eiichi,Shibata, Daisuke,Doi, Etsushiro
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p. 2439 - 2446
(2007/10/02)
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- Peptide Synthesis by Means of Immobilized Enzymes II. Immobilized Trypsin, Thermolysin and Papain
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Model studies were performed on the utility of covalently immobilized trypsin, thermolysin and papain for peptide bond formation.Trypsin and thermolysin catalyzed the formation of peptide bonds with nearly the same efficiency as the soluble proteases and they could be re-used successfully for further coupling experiments.The possibility of using immobilized trypsin and papain for kinetically controlled peptide bond formation was investigated.With the serine type enzyme trypsin excellent product yields were obtained starting with ester carboxyl components and an economical ratio of substrates.Experiments with the thiol protease papain were unsatisfactory because the once formed product is hydrolyzed as fact as the starting ester substrate used. - Keywords: Immobilized enzymes; Papain; Peptide synthesis; Thermolysin; Trypsin
- Koennecke, Andreas,Haensler, Marion,Schellenberger, Volker,Jakubke, Hans-Dieter
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p. 433 - 444
(2007/10/02)
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- MONITORED AMINOLYSIS OF 3-ACYL-1,3-THIAZOLIDINE-2-THIONE WITH AMINO ACID AND ITS DERIVATIVE: PEPTIDE BOND FORMATION, CHEMOSELECTIVE ACYLATION, AND BRIDGING REACTION
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As a new extention of the monitored aminolysis of 3-acyl-1,3-thiazolidine-2-thione, its applications to peptide bond formation, chemoselective acylation of amino acid, and bridging reaction on the enzyme model are reported.
- Nagao, Yoshimitsu,Miyasaka, Tadayo,Seno, Kaoru,Yagi, Masahiro,Fujita, Eiichi
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p. 463 - 466
(2007/10/02)
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