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1.
Myoglobin imprinted in aqueous solution with ligands binding to its heme iron, followed by lyophilization, catalyzed the epoxidation of styrene with H2O2or tert-butyl hydroperoxide in organic solvents much faster than the non-imprinted protein under otherwise the same conditions. 相似文献
2.
Stéphane Roux Elisabeth Zékri Bernard Rousseau Maïté Paternostre Jean‐Christophe Cintrat Nicolas Fay 《Journal of peptide science》2008,14(3):354-359
Most synthesized peptides are nowadays produced using solid-phase procedures. Due to cleavage and purification conditions, they are mainly obtained in the presence of trifluoroacetic acid (TFA) and, for cationic peptides, as trifluoroacetate (TF-acetate) salts. However, TF-acetate interferes with physicochemical characterizations using infrared spectroscopy and might significantly affect the in vivo studies. Thus, TF-acetate exchange by another counter-ion is often required. Up to now, the classical procedure has consisted of freeze-drying the peptide several times in the presence of an excess of a stronger acid than TFA (pKa approximately 0): generally HCl (pKa = - 7). This approach means that working at pH < 1 can induce peptide degradation. We therefore tested three different approaches to exchange the tightly bound TF-acetate counter-ion from the dicationic octapeptide lanreotide: (i) reverse-phase HPLC, (ii) ion-exchange resin, and (iii) deprotonation/reprotonation cycle of the amino groups. The first two approaches allow the partial to almost complete exchange of the TF-acetate counter-ion by another ion from an acid weaker than TFA, such as acetic acid (pKa = 4.5), and the third requires a basic solution that permits the complete removal of TF-acetate counter-ion. The efficiency of these three procedures was tested and compared by using different analytical techniques such as 19F-NMR, 1H-NMR and attenuated total reflectance Fourier transformed infrared spectroscopy (ATR FT-IR). We also show that ATR-IR can be used to monitor the TFA removal. The counter-ion exchange procedures described in this study are easy to carry out, fast, harmless and reproducible. Moreover, two of them offer the very interesting possibility of exchanging the initial TF-acetate by any other counter-ion. 相似文献
3.
Mahesh S. Chandak Takashi Nakamura Toshio Takenaka Tapan K. Chaudhuri Maho Yagi‐Utsumi Jin Chen Koichi Kato Kunihiro Kuwajima 《Protein science : a publication of the Protein Society》2013,22(4):486-491
Dimethylsulfoxide (DMSO)‐quenched hydrogen/deuterium (H/D)‐exchange is a powerful method to characterize the H/D‐exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non‐protected fast‐exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO‐quenched H/D‐exchange studies of proteins so far reported, lyophilization was used to remove D2O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two‐dimensional nuclear magnetic resonance (2D NMR) spectra. The denaturants or salts remaining after lyophilization thus prevent the measurement of good NMR spectra. In this article, we report that the use of spin desalting columns is a very effective alternative to lyophilization for the medium exchange from the D2O buffer to the DMSO solution. We show that the medium exchange by a spin desalting column takes only about 10 min in contrast to an overnight length of time required for lyophilization, and that the use of spin desalting columns has made it possible to monitor the H/D‐exchange behavior of a fully unfolded protein in a concentrated denaturant. We report the results of unfolded ubiquitin in 6.0M guanidinium chloride. 相似文献
4.
Ishimaru Masanori Yoshizawa-Kumagaye Kumiko Kubo Shigeru Kitani Tetsuya Chino Naoyoshi- Kangawa Kenji Kimura Terutoshi 《International journal of peptide research and therapeutics》2003,10(1):41-50
Summary Rat ghrelin, a 28-amino acid residue peptide with an octanoyl group at the side chain of Ser3, was synthesized chemically
by applying Fmoc/
t
Bu strategy. An ester linkage between octanoic acid and the hydroxyl function of Ser3 was found to be maintained without serious
damage during the final deprotection with trifluoroacetic acid (TFA). The most notable finding was the counter-ion-dependent
stability change of the octanoyl moiety in the molecule. After consolidation of the counter-ion to TFA (TFA form), the octanoyl
group persisted stably upon dissolution in water, whereas in the case of the acetate-form peptide, both de-octanoylation and
dehydration (formation of the dehydro-Ala residue) occurred in aqueous solution at the same Ser3 residue. The amounts of these
degraded products varied with factors such as solvent, temperature and times of lyophilization. These experimental findings
lay the basis for performing the bioassay of ghrelin, which has an octanoyl moiety involved in its numerous biological activities
thus far revealed. 相似文献
5.
Masanori Ishimaru Kumiko Yoshizawa-Kumagaye Shigeru Kubo Tetsuya Kitani Naoyoshi- Chino Kenji Kangawa Terutoshi Kimura 《Letters in Peptide Science》2003,10(1):41-50
Rat ghrelin, a 28-amino acid residue peptide with an octanoyl group at the side chain of Ser3, was synthesized chemically by applying Fmoc/tBu strategy. An ester linkage between octanoic acid and the hydroxyl function of Ser3 was found to be maintained without serious damage during the final deprotection with trifluoroacetic acid (TFA). The most notable finding was the counter-ion-dependent stability change of the octanoyl moiety in the molecule. After consolidation of the counter-ion to TFA (TFA form), the octanoyl group persisted stably upon dissolution in water, whereas in the case of the acetate-form peptide, both de-octanoylation and dehydration (formation of the dehydro-Ala residue) occurred in aqueous solution at the same Ser3 residue. The amounts of these degraded products varied with factors such as solvent, temperature and times of lyophilization. These experimental findings lay the basis for performing the bioassay of ghrelin, which has an octanoyl moiety involved in its numerous biological activities thus far revealed. 相似文献
6.
A sequential extraction scheme was combined with sorption isotherm analysis in order to investigate sorption of sewage sludge-derived Cu and Zn to the A-horizon of a humic-gley soil as a whole, and to the operationally defined exchangeable (1?M MgCl2), carbonate (1?M NaOAc), Fe/Mn oxide (0.04?M NH2OH.HCl), and organic (0.02?M HNO3+30% H2O2) soil fractions. Sorption parameters were compared for a sample of sludge leachate (with 97.4% of Cu and 63.2% of Zn present as dissolved metal-organic matter complexes, as calculated by geochemical modeling involving MINTEQA2 and verified using an ion exchange resin method) with that of a reference solution exhibiting the same chemical characteristics as the leachate, except for the presence of dissolved organic material. Dissolved metal-organic matter complexes were found to significantly (P<0.05) depress sorption to the bulk soil and each fraction. The greatest depression of Cu and Zn sorption was observed for the exchangeable, carbonate, and Fe/Mn oxide fractions, while the organic fraction of the soil was the least affected. This reflects a greater affinity for the exchangeable, carbonate, and Fe/Mn oxide fractions by the free divalent metal (Cu2+, Zn2+), with sorption by these fractions attributed to cation exchange, chemisorption, and co-precipitation processes. The sorption characteristics of the organic fraction indicated that Cu and Zn sorption by soil organic matter mostly involved dissolved metal-organic matter complexes. This may be attributed to hydrophobic interactions between nonpolar regions of the dissolved metal-organic matter complexes and solid-phase soil organic matter. 相似文献
7.
Trifluoroacetate (CF3COO-, or TFA) is almost always present in commercially synthesized peptides. Unfortunately, it has a strong infrared (IR) absorption band at 1673 cm-1, significantly overlapping or even completely obscuring the amide I band of a peptide. In such cases TFA must be removed from the solution in order to be able to use IR absorption spectroscopy for peptide secondary structure determination. The most convenient and widely used procedure involves peptide lyophilization from a 0.1 M HCl solution. In our studies of the tryptophan-rich antimicrobial peptide indolicidin, we have found that caution should be taken when using this HCl concentration. High HCl concentrations (>10 mM in unbuffered solutions and > 50 mM in buffered solutions) may modify the peptide structure and reduce its thermal stability, thereby interfering with subsequent structural investigations of the peptide. Our results indicate that HCl concentrations between 2 and 10 mM are adequate to remove essentially all TFA impurities without any modification of the peptide secondary structure. 相似文献
8.
Effects of Polar Organic Solvents on the Biocatalysis of Chlorophyllase in a Biphasic Organic System
《Bioscience, biotechnology, and biochemistry》2013,77(11):1947-1952
The effects of polarity of various organic solvents, including acetone, ethanol, and propanol, used in a biphasic organic system, on the hydrolytic activity of a partially purified chlorophyllase from Phaeodactylum tricornutum were investigated. The different concentrations of each polar organic solvent, from 0 to 40%, were added to a mixture (45:55, v/v) of hexane and a buffer solution of Tris–HCl (20 mm, pH 7.5). The most appropriate concentrations of acetone, ethanol, and propanol for the hydrolytic activity of chlorophyllase were 12.5, 5.0, and 2.5%, respectively. The results indicated that the optimum reaction time for the chlorophyllase activity in the biphasic system decreased from 7.0 h to 3.0, 5.0, and 5.0 h, respectively, upon the addition of an appropriate amount of acetone, ethanol, or propanol. The Vmax and Km as well as the inhibitory effect of phytol on the chlorophyllase activity in the biphasic organic system containing a polar organic solvent were also investigated. 相似文献
9.
Chieh‐Ming J. Chang Yung‐Chuan Liu Wen‐Dee Chiang Chwen‐Jen Shieh 《Biotechnology progress》2012,28(6):1443-1449
A peptide, N‐Ac‐Phe‐Tyr‐NH2, with angiotensin I‐converting enzyme (ACE) inhibitor activity was synthesized by an α‐chymotrypsin‐catalyzed condensation reaction of N‐acetyl phenylalanine ethyl ester (N‐Ac‐Phe‐OEt) and tyrosinamide (Tyr‐NH2). Three kinds of solvents: a Tris–HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic‐aqueous solvent (Tris‐HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N‐Ac‐Phe‐Tyr‐NH2 could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N‐Ac‐Phe‐Tyr‐NH2, so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012 相似文献
10.
CD study of four modified nucleosides, constituents of tRNA molecules, revealed that 2-thio-5-methyluridine and 5-methyluridine in aqueous solution, 0.1N HCl, and organic solvents essentially occur in an anti-conformation. 5-Methylcytidine also occurs in an anti-conformation similar to cytidine in aqueous solution and organic solvents, while 2-thiocytidine dihydrate appears to occur in an anti-conformation. It is stressed that the CD data of thionucleosides might be applied to the successfully conformational analysis of tRNA molecules. 相似文献
11.
《Journal of biotechnology》1988,7(2):141-146
A magnetic fluid was synthesized by oxidation of ferrous ions (Fe2&+) in the presence of a synthetic alternating copolymer of polyethylene glycol (PEG) and maleic acid (MA), poly(PEG-MA). The magnetic fluid dispersed stably both in aqueous solution and in organic solvents. Its particle size was approximately 10 nm. The magnetic fluid was mixed with lipase in water, followed by lyophilization. Although the enzyme and the magnetic fluid were dissociated in aqueous solution, they remained associated in organic solvents such as benzene. The magnetic fluid-adsorbed lipase dispersed in benzene and exerted high enzymic activity (2.9 μmol min−1 mg−1 lyophilized powder) for lauryl laurate synthesis from lauric acid and lauryl alcohol, and was readily recovered from the reaction mixture in a magnetic field (6000 Oe) without loss of enzymic activity. 相似文献
12.
When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N-Ac-L-Phe-NH(2), their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand-induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramaticaliy enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggesting the same mechanism of action. Excipient activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient-induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its "regular" counterpart. (c) 1993 John Wiley & Sons, Inc. 相似文献
13.
Viviane Forsberg Renyun Zhang Joakim B?ckstr?m Christina Dahlstr?m Britta Andres Magnus Norgren Mattias Andersson Magnus Hummelg?rd H?kan Olin 《PloS one》2016,11(4)
Many solution processing methods of exfoliation of layered materials have been studied during the last few years; most of them are based on organic solvents or rely on surfactants and other funtionalization agents. Pure water should be an ideal solvent, however, it is generally believed, based on solubility theories that stable dispersions of water could not be achieved and systematic studies are lacking. Here we describe the use of water as a solvent and the stabilization process involved therein. We introduce an exfoliation method of molybdenum disulfide (MoS2) in pure water at high concentration (i.e., 0.14 ± 0.01 g L−1). This was achieved by thinning the bulk MoS2 by mechanical exfoliation between sand papers and dispersing it by liquid exfoliation through probe sonication in water. We observed thin MoS2 nanosheets in water characterized by TEM, AFM and SEM images. The dimensions of the nanosheets were around 200 nm, the same range obtained in organic solvents. Electrophoretic mobility measurements indicated that electrical charges may be responsible for the stabilization of the dispersions. A probability decay equation was proposed to compare the stability of these dispersions with the ones reported in the literature. Water can be used as a solvent to disperse nanosheets and although the stability of the dispersions may not be as high as in organic solvents, the present method could be employed for a number of applications where the dispersions can be produced on site and organic solvents are not desirable. 相似文献
14.
《Journal of Molecular Catalysis .B, Enzymatic》2011,72(3-4):171-176
Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion bodies can be successfully refolded. In this study, the different parameters were investigated and optimized on the refolding of denatured lipase. The maximum lipase activity of 5000 U/L was obtained after incubation of denatured enzyme in a refolding buffer containing 20 mM Tris–HCl (pH 7.0), 1 mM Ca2+ at 20 °C. Then, the refolded lipase was purified to homogeneity by anion exchange chromatography. The purified refolded lipase was stable in broad ranges of temperatures and pH values, as well as in a series of water-miscible organic solvents. In addition, some water-immiscible organic solvents, such as petroleum ether and isopropyl ether, could reduce the polarity and increase the nonpolarity of the refolding system. The results of Fourier transform infrared (FT-IR) microspectroscopy were the first to confirm that lipase refolding could be further improved in the presence of organic solvents. The purified refolded lipase could enantioselectively hydrolyze trans-3-(4-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM]. These features render the lipase attraction for biotechnological applications in the field of organic synthesis and pharmaceutical industry. 相似文献
15.
Oligopeptides having the general formula N-carbobenzoxy(γ-methyl-L -glutamyl)n-dimethyl-L -glutamate (with n = 1, 3, 5, 7, 11) were prepared, using both solid-phase synthesis and conventional peptide couplings in solution. The appearance of an ordered structure with increasing chain length was studied in organic solvents and at the air–water interface. The results obtained by thin-layer chromatography were interpreted. All the studies have demonstrated a very similar behaviour of this type of oligopeptide in solution and in monolayers. 相似文献
16.
Cherl-Ho Lee Shek Kwan Tsang Rintaro Urakabe Cho Kyun Rha 《Biotechnology and bioengineering》1979,21(1):1-17
The morphology of dried Candida lipolytica yeast suspended in aqueous solutions (H2O, 0.4% NaOH, 2N HCl, and 6N HCl) and organic solvents (95% alcohol and acetone) was studied using a scanning electron microscope (SEM) and an optical microscope. The effect of high-pressure homogenization on cell-wall structure and cell clumps was also determined. The protein extractability, sedimentation property, and viscosity of cells subjected to different mechanical and chemical treatments were also investigaged. The dried yeast cells were in a spherical agglomeration consisting of 100s of closely bound cells. The clump was resistant to water, aqueous 2N HCl solution at 25°C, 95% alcohol and acetone, but vulnerable to 6N HCl, aqueous 0.4% NaOH solution, and homogenization. The homogenization of the cell suspension not only broke the clump but also cracked the cell-wall structure. The aqueous alkaline solution could have weakened the cell wall and increased the solubility of the protein released through the cracks in the cell wall. The destruction of the agglomeration and the cell-wall structure increased the hydration of the cell and thereby increased the stability of the suspension. The sedimentation and the viscosity of the cell suspension corresponded to the morphological changes and the extractability of protein in the cell suspensions with different treatments. 相似文献
17.
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
18.
The chemoenzymatic epoxidation of a terpene alcohol, citronellol, is reported. Some experimental conditions, such as the use of lipases from different sources, oxidizing agents (H2O2 or urea–hydrogen peroxide, UHP), reaction time, acyl donor type (C6–C16), temperature (15–40 °C) and the influence of organic media, were evaluated. In most cases, citronellol oxide 2 or the ester citronellol oxide 3 were obtained. Depending on the reaction conditions, high yields of products 2 or 3 were obtained (>99%). CAL-B was the most effective catalyst in this reaction. For epoxide 2, the highest yields of 80% and 77% were obtained at 20 °C and 25 °C, respectively, using UHP as an oxidizing agent and octanoic acid as an acyl donor. The organic medium appears to be one of the most important parameters in the reaction. Using chloroform or dichloromethane, product 2 was obtained at a >99% yield after 24 h. When different mixtures consisting of varied organic solvents and an imidazolium-based ionic liquid (IL) were used, the results were dependent on both the solvent and IL counter-ion (18–75%). 相似文献
19.
Halohydrin dehalogenase HheG is an industrially interesting biocatalyst for the preparation of different β-substituted alcohols starting from bulky internal epoxides. We previously demonstrated that the immobilization of different HheG variants in the form of cross-linked enzyme crystals (CLECs) yielded stable and reusable enzyme immobilizes with increased resistance regarding temperature, pH, and the presence of organic solvents. Now, to further establish their preparative applicability, HheG D114C CLECs cross-linked with bis-maleimidoethane have been successfully produced on a larger scale using a stirred crystallization approach, and their application in different chemical reactor types (stirred tank reactor, fluidized bed reactor, and packed bed reactor) was systematically studied and compared for the ring opening of cyclohexene oxide with azide. This revealed the highest obtained space-time yield of 23.9 kgproduct gCLEC−1 h−1 Lreactor volume−1 along with the highest achieved product enantiomeric excess [64%] for application in a packed-bed reactor. Additionally, lyophilization of those CLECs yielded a storage-stable HheG preparation that still retained 67% of initial activity (after lyophilization) after 6 months of storage at room temperature. 相似文献
20.
C. Tsitsimpikou D. Voutou P. Christakopoulos D. Kekos B. J. Macris F. N. Kolisis 《Biotechnology letters》1994,16(1):57-62
Summary The effect of organic solvents on the stability of -glucosidase in a powder form, isolated fromFusarium oxysporum, has been studied using several organic solvents of different degree of hydrophobicity. It was found that -glucosidase remains quite stable after a prolonged incubation in the presence of most of the organic solvents used, even at temperatures as high as 70°C. Only dimethyformamide (DMF) and tetrahydrofuran (THF) reduce considerably the enzyme activity in a short preincubation period. Studies on the effect of the pH of the buffer used prior to lyophilization, as well as of exogenous added water to the incubation mixture, on enzyme stability show that it is more stable in pH 5.0 and in the lowest water content. In addition it was found that the presence of glucose in the lyophilization procedure gives a significant protection to the enzyme when it is incubated for 30 h in pentanol and n-hexane. 相似文献