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1.
Circular dichroism spectroscopy was used to study the effect of l-arginine on the temperature related unfolding and aggregation of three growth hormones, i.e. human, porcine and mink growth hormones, and human interferon-α2b. l-arginine can stabilize some proteins and suppress their aggregation as it was exemplified by porcine and mink growth hormones. For some other proteins, on the contrary, the effect of arginine can be negative. Even at low concentrations the amino acid is able to promote the aggregation as it was demonstrated by the experiments with human growth hormone and interferon-α2b. l-arginine seems not to be a universal excipient for preventing the temperature related aggregation of proteins in contrast to its widespread application in the refolding process. 相似文献
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3.
l ‐Cysteine is widely used as a precursor in the pharmaceutical, cosmetic, food, and feed additive industries. It has been industrially produced from hydrolysis of human and animal hairs, which is limited for industrial production. At the same time, chemical hydrolysis causes the formation of intractable waste material. Thus, environmentally friendly methods have been developed. A big obstacle of currently available methods is the low substrate solubility leading to poor l ‐cysteine yield. Here, a method for improving the low solubility of the substrate d ,l ‐2‐amino‐Δ2‐thiazoline‐4‐carboxylic acid (d ,l ‐ATC) is presented and the enzymatic reaction at high concentration levels was optimized. The substrate was dissolved in large amounts in aqueous solutions by pH control using salts. d ,l ‐ATC solubility increased with an increasing solution pH due to its enhanced hydrophilicity, which can be achieved by a shift to dissociated carboxylic group (–COO?). The highest d ,l ‐ATC solubility of 610 mM was obtained at pH 10.5. The maximum l ‐cysteine yield of 250 mM was attained at pH 9.1, which lies between the optimum values for high substrate solubility and reaction rate. The product yield could be increased by more than 10 times compared to those in previous reports, which is industrially meaningful. 相似文献
4.
l ‐Arginine (Arg), l ‐homoarginine (HArg), l ‐arginine ethylester (ArgEE), and l ‐arginine methylester (ArgME) were found effective in inhibiting protein aggregation, but the molecular mechanisms are not clear. Herein, stopped‐flow fluorescence spectroscopy, isothermal titration calorimetry, and mass spectroscopy were used to investigate the folding kinetics of lysozyme and the interactions of the additives with lysozyme. It was found that the interactions of ArgME and ArgEE with lysozyme were similar to that of guanidine hydrochloride and were much stronger than those of Arg and HArg. The binding forces were all mainly hydrogen bonding and cation‐π interaction from the guanidinium group, but their differences in molecular states led to the significantly different binding strengths. The additives formed molecular clusters in an increasing order of ArgEE, ArgME, HArg, and Arg. Arg and HArg mainly formed annular clusters with head‐to‐tail bonding, while ArgME and ArgEE formed linear clusters with guanidinium groups stacking. The interactions between the additives and lysozyme were positively related to the monomer contents. That is, the monomers were the primary species that participated in the direct interactions due to their intact guanidinium groups and small sizes, while the clusters performed as barriers to crowd out the protein–protein interactions for aggregation. Thus, it is concluded that the effects of Arg and its derivatives on protein aggregation stemmed from the direct interactions by the monomers and the crowding effects by the clusters. Interplay of the two effects led to the differences in their inhibition effects on protein aggregation. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1316–1324, 2013 相似文献
5.
l-Arginine was used to suppress the aggregation of recombinant mink and porcine growth hormones in the refolding process from
E. coli inclusion bodies by solubilization–dilution protocol at high protein concentration and pH 8.0. The influence of l-arginine concentration on the renaturation yield of both proteins was investigated. l-Arginine effectively suppressed the precipitation of growth hormones during dilution, but did not inhibit soluble oligomers
formation. The results of mink and porcine growth hormones purification from 4 g of biomass are presented. 相似文献
6.
The influence of l-arginine on the temperature-induced aggregation of porcine and mink growth hormones was studied by fluorescence spectroscopy.
It was found that l-arginine suppresses the heat-induced aggregation. Moreover, the analysis of l-arginine interaction with the native proteins by fluorescence spectroscopy and circular dichroism spectroscopy revealed no
significant changes in their native structure. On the basis of the results, l-arginine could be considered as a potential additive for the prevention of storage and temperature-related denaturation and
aggregation of veterinary growth hormones. 相似文献
7.
Egle Bajorunaite Andrejus Cirkovas Kostas Radzevicius Kim Lambertsen Larsen Jolanta Sereikaite Vladas-Algirdas Bumelis 《International journal of biological macromolecules》2009,44(5):428-434
Cyclodextrins with different ring size and ring substituents were tested for recombinant mink and porcine growth hormones aggregation suppression in the refolding process from Escherichia coli inclusion bodies. Methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin show a positive effect on the aggregation suppression of both proteins. The influence of different methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin concentrations on the renaturation yield of both growth hormones was investigated. Moreover, methyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin suppress not only folding-related, but also temperature-related aggregates formation of both proteins. Circular dichroism experiments (monitoring of protein solution turbidity by registering high tension voltage) showed that the onset temperature of aggregation of both growth hormones increased with increasing 2-hydroxypropyl-β-cyclodextrin concentration. In conclusion, cyclodextrins have perspectives in biotechnology of veterinary growth hormones not only for protein production, but also for its storage. 相似文献
8.
In the past years, enantioanalysis became very important for clinical analysis; biomarkers/substances of biomedical importance with chiral structure should be analyzed and their presence correlated with the specific disorder. Therefore, we developed a method for the assay of l ‐ and d ‐glucose, based on molecular recognition of l ‐ and d ‐glucose. While for d ‐glucose there are many methods to assess its quantity, the l ‐enantiomer is not routinely detected by standard methods. Two stochastic microsensors based on the immobilization of Copper(II)phthalocyanine and Ni(II)phthalocyanine, in natural diamond powder, were proposed for the enantioanalysis of glucose. The proposed methods proved to have high sensitivities and were able to be used for determination of concentrations as low as 2.5 pg mL?1 for d ‐glucose and as low as 2.5 fg mL?1 for l ‐glucose. The enatioanalysis was performed with good results in whole blood samples collected from diabetic patients. 相似文献
9.
Cysteine inhibits amyloid fibrillation of lysozyme and directs the formation of small worm‐like aggregates through non‐covalent interactions
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Eisuke Takai Ken Uda Shuhei Matsushita Yui Shikiya Yoichi Yamada Kentaro Shiraki Tamotsu Zako Mizuo Maeda 《Biotechnology progress》2014,30(2):470-478
In this article, we discuss the effects of amino acids on amyloid aggregation of lysozyme. l ‐cysteine (Cys) dramatically inhibited fibrillation of lysozyme, whereas other amino acids (including l ‐arginine) did not. In the presence of Cys, the aggregation pathway of lysozyme shifted from fibrillation to the formation of the small worm‐like aggregates with unfolding. The interaction between Cys and lysozyme was observed to be non‐covalent, suggesting that the thiophilic interaction between the thiol group on the side chain of Cys and the core sequence of lysozyme significantly contributes to the inhibition of amyloid aggregation. These findings provide a new basis for the design of a biocompatible additive to prevent amyloid fibrillation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:470–478, 2014 相似文献
10.
L ‐Homophenylalanine (L ‐HPA) and N6‐protected‐2‐oxo‐6‐amino‐hexanoic acid (N6‐protected‐OAHA) can be used as building blocks for the manufacture of angiotensin‐converting enzyme inhibitors. To synthesize L ‐HPA and N6‐protected‐OAHA simultaneously from 2‐oxo‐4‐phenylbutanoic acid (OPBA) and N6‐protected‐L ‐lysine, several variants of Escherichia coli aspartate aminotransferase (AAT) were developed by site‐directed mutagenesis and their catalytic activities were investigated. Three kinds of N6‐protected‐L ‐lysine were tested as potential amino donors for the bioconversion process. AAT variants of R292E/L18H and R292E/L18T exhibited specific activities of 0.70±0.01 U/mg protein and 0.67±0.02 U/mg protein to 2‐amino‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐lysine) and 2‐amino‐6‐(2,2,2‐trifluoro‐acetylamino)‐hexanoic acid, respectively. E. coli cells expressing R292E/L18H variant were able to convert OPBA and BOC‐lysine to L ‐HPA and 2‐oxo‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐OAHA) with 96.2% yield in 8 h. This is the first report demonstrating a process for the simultaneous production of two useful building blocks, L ‐HPA and BOC‐OAHA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
11.
Lúcia F. A. Amorim Fani Sousa Joo A. Queiroz Carla Cruz ngela Sousa 《Journal of molecular recognition : JMR》2015,28(6):349-358
The growing demand of pharmaceutical‐grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l ‐histidine and its derivatives, 1‐benzyl‐l ‐histidine and 1‐methyl‐l ‐histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l ‐histidine and its derivatives was high (KD > 10−8 M), and the highest affinity was found for human papillomavirus 16 E6/E7 (KD = 1.1 × 10−10 M and KD = 3.34 × 10−10 M for open circular and linear plasmid isoforms, respectively). l ‐Histidine and 1‐benzyl‐l ‐histidine were immobilized on monolithic matrices. Chromatographic studies of l ‐histidine and 1‐benzyl‐l ‐histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1‐benzyl‐l ‐histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l ‐histidine monolith was 29‐fold higher than that obtained with conventional l ‐histidine agarose. Overall, the combination of either l ‐histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications. Copyright © 2015 John Wiley & Sons, Ltd. 相似文献
12.
Improved ε‐poly‐l‐lysine productivity partly resulting from rapid cell growth in cultures using a glucose–glycerol mixed carbon source
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Xin Zeng Xu‐Sheng Chen Xi‐Dong Ren Liang Wang Yang Gao Zhong‐Gui Mao 《Engineering in Life Science》2016,16(5):443-452
Productivity enhancements with mixed carbon sources are usually accompanied by simultaneous improvement of cell growth. However, whether the enhanced cell growth in mixed carbon sources influences the biochemical productivity of ε‐poly‐l ‐lysine (ε‐PL) still remains unclear. In this study, we investigated the effect of growth rate on the ε‐PL productivity in a glucose–glycerol mixed carbon source. Based on the typical ε‐PL fed batch fermentation, chemostat culture and relevant physiological analyses were carried out. The ε‐PL productivity was positively correlated to the growth rate ranging from 0.02 to 0.06 h?1. The primary metabolism activity was enhanced at higher growth rate, providing sufficient precursor l ‐lysine and energy for ε‐PL production. Meanwhile, these two key elements were equally important for biomass production, which could be quickly produced when the cells were fast growing. In addition, rapid growth also strengthened the antioxidant capacity of cells to defend potential oxidative stress. The positive correlation between the growth rate and ε‐PL productivity indicated that the improvement of ε‐PL productivity in the mixed carbon source was partly attributed to the simultaneously enhanced cell growth. Information obtained may provide references for further studies on other secondary biochemicals’ production using mixed carbon sources. 相似文献
13.
Induction of systemic disease resistance in Nicotiana benthamiana by the cyclodipeptides cyclo (l‐Pro‐l‐Pro) and cyclo (d‐Pro‐d‐Pro)
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Cyclodipeptides, formed from two amino acids by cyclodehydration, are produced naturally by many organisms, and are known to possess a large number of biological activities. In this study, we found that cyclo (l ‐Pro‐l ‐Pro) and cyclo (d ‐Pro‐d ‐Pro) (where Pro is proline) could induce defence responses and systemic resistance in Nicotiana benthamiana. Treatment with the two cyclodipeptides led to a reduction in disease severity by Phytophthora nicotianae and Tobacco mosaic virus (TMV) infections compared with controls. Both cyclopeptides triggered stomatal closure, induced reactive oxygen species production and stimulated cytosolic calcium ion and nitric oxide production in guard cells. In addition, the application of cyclodipeptides significantly up‐regulated the expression of the plant defence gene PR‐1a and the PR‐1a protein, and increased cellular salicylic acid (SA) levels. These results suggest that the SA‐dependent defence pathway is involved in cyclodipeptide‐mediated pathogen resistance in N. benthamiana. We report the systemic resistance induced by cyclodipeptides, which sheds light on the potential of cyclodipeptides for the control of plant diseases. 相似文献
14.
Cassandra M. Joiner Meghan E. Breen Anna K. Mapp 《Protein science : a publication of the Protein Society》2019,28(6):1163-1170
The photoactivatable amino acid p‐benzoyl‐l ‐phenylalanine (pBpa) has been used for the covalent capture of protein–protein interactions (PPIs) in vitro and in living cells. However, this technique often suffers from poor photocrosslinking yields due to the low reactivity of the active species. Here we demonstrate that the incorporation of halogenated pBpa analogs into proteins leads to increased crosslinking yields for protein–protein interactions. The analogs can be incorporated into live yeast and upon irradiation capture endogenous PPIs. Halogenated pBpas will extend the scope of PPIs that can be captured and expand the toolbox for mapping PPIs in their native environment. 相似文献
15.
Analysis of intracellular metabolites of Corynebacterium glutamicum at high cell density with automated sampling and filtration and assessment of engineered enzymes for effective l‐lysine production
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Julian A. da Luz Enrico Hans Doinita Frank An‐Ping Zeng 《Engineering in Life Science》2017,17(5):512-522
Engineering of enzymes and pathways is generally required for the development of efficient strains for bioproduction processes. To this end, quantitative and reliable data of intracellular metabolites are highly desired, but often not available, especially for conditions more close to industrial applications, i.e. at high cell density and product concentration. Here, we investigated the intracellular metabolite profiles of an engineered l ‐lysine‐producing Corynebacterium glutamicum strain and the corresponding wild‐type strain to assess the impacts of deregulation of product inhibition of the key enzymes aspartate kinase and phosphoenolpyruvate carboxylase and to identify potentials for their further improvement. A bioreactor system with automated fast‐sampling, filtration and on‐filter quenching of the metabolism was used for a more reliable determination of intracellular metabolites in batch cultures with optical cell density (OD660) up to 40. The l ‐lysine‐producing strain showed substantially different metabolite profiles in the amino acid metabolism, including increased intracellular pool sizes in the l ‐lysine‐, l ‐homoserine‐ and l ‐threonine pathways and decreased intracellular pool sizes for all other determined amino acids. By comparing data of in vitro inhibition of the engineered enzymes and determined intracellular concentrations of the inhibitors it was found that the inferred in vivo activities of these enzymes are still significantly below their in vitro maximums. This work demonstrates the usefulness of metabolic analysis for assessing the impact of engineered enzymes and identifying targets for further strain development. 相似文献
16.
On the Dynamical Behavior of the Cysteine Dioxygenase‐l‐Cysteine Complex in the Presence of Free Dioxygen and l‐Cysteine
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Francesco Pietra 《化学与生物多样性》2017,14(11)
In this work, viable models of cysteine dioxygenase (CDO) and its complex with l ‐cysteine dianion were built for the first time, under strict adherence to the crystal structure from X‐ray diffraction studies, for all atom molecular dynamics (MD). Based on the CHARMM36 FF, the active site, featuring an octahedral dummy Fe(II) model, allowed us observing water exchange, which would have escaped attention with the more popular bonded models. Free dioxygen (O2) and l ‐cysteine, added at the active site, could be observed being expelled toward the solvating medium under Random Accelerated Molecular Dynamics (RAMD) along major and minor pathways. Correspondingly, free dioxygen (O2), added to the solvating medium, could be observed to follow the same above pathways in getting to the active site under unbiased MD. For the bulky l ‐cysteine, 600 ns of trajectory were insufficient for protein penetration, and the molecule was stuck at the protein borders. These models pave the way to free energy studies of ligand associations, devised to better clarify how this cardinal enzyme behaves in human metabolism. 相似文献
17.
Five compounds of different chemical structure were tested for aggregation suppression during the refolding of porcine and mink growth hormones as model proteins from Escherichia coli inclusion bodies by the dilution method. Of all compounds tested in this work, 3-guanidinopropionic acid (GPA) containing a guanidinium group was the most effective additive for aggregation suppression. Anti-aggregatory properties of GPA were compared with the ones of l-arginine. 相似文献
18.
Y.‐R. Lim R.‐Y. Yoon E.‐S. Seo Y.‐S. Kim C.‐S. Park D.‐K. Oh 《Journal of applied microbiology》2010,109(4):1188-1197
Aims: To characterize of a thermostable recombinant α‐l ‐arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino‐oligosaccharides to l ‐arabinose. Methods and Results: A recombinant α‐l ‐arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi‐Trap anion exchange chromatography with a specific activity of 28·2 U mg?1. The native enzyme was a 58‐kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α‐l ‐arabinofuranosidases were completely conserved in α‐l ‐arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5·5 and 80°C with a half‐life of 49 h at 75°C. Among aryl‐glycoside substrates, the enzyme displayed activity only for p‐nitrophenyl‐α‐l ‐arabinofuranoside [maximum kcat/Km of 220 m(mol l?1)?1 s?1] and p‐nitrophenyl‐α‐l ‐arabinopyranoside. This substrate specificity differs from those of other α‐l ‐arabinofuranosidases. In a 1 mmol l?1 solution of each sugar, arabino‐oligosaccharides with 2–5 monomer units were completely hydrolysed to l ‐arabinose within 13 h in the presence of 30 U ml?1 of enzyme at 75°C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino‐oligosaccharides of α‐l ‐arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l ‐arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino‐oligosaccharides performed by thermostable α‐l ‐arabinofuranosidase. 相似文献
19.
Shuai Qiu Ranran Zhu Yanyan Zhao Xiaoping An Fengjing Jia Jinxiu Peng Zelin Ma Yuanyuan Zhu Jiayi Wang Jinhuan Su Qingjun Wang Hailin Wang Yuan Li Kairong Wang Wenjin Yan Rui Wang 《Journal of peptide science》2017,23(5):392-402
The misuse and overuse of antibiotics result in the emergence of resistant bacteria and fungi, which make an urgent need of the new antimicrobial agents. Nowadays, antimicrobial peptides have attracted great attention of researchers. However, the low physiological stability in biological system limits the application of naturally occurring antimicrobial peptides as novel therapeutics. In the present study, we synthesized derivatives of protonectin by substituting all the amino acid residues or the cationic lysine residue with the corresponding D ‐amino acids. Both the D ‐enantiomer of protonectin (D ‐prt) and D ‐Lys‐protonectin (D ‐Lys‐prt) exhibited strong antimicrobial activity against bacteria and fungi. Moreover, D ‐prt showed strong stability against trypsin, chymotrypsin and the human serum, while D ‐Lys‐prt only showed strong stability against trypsin. Circular dichroism analysis revealed that D ‐Lys‐prt still kept typical α‐helical structure in the membrane mimicking environment, while D ‐prt showed left hand α‐helical structure. In addition, propidium iodide uptake assay and bacteria and fungi killing experiments indicated that all D ‐amino acid substitution or partially D ‐amino acid substitution analogs could disrupt the integrity of membrane and lead the cell death. In summary, these findings suggested that D ‐prt and D ‐Lys‐prt might be promising candidate antibiotic agents for therapeutic application against resistant bacteria and fungi infection. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
20.
Sergey Nikitin Daniel Palmer Morten Meldal Frederik Diness 《Journal of peptide science》2016,22(10):642-646
Detailed studies comparing solid‐supported l ‐ or d ‐amino acid adhesion peptides based on the sequence KLHRIRA were performed. Stability towards proteases and levels of cellular adhesion to the otherwise inert surface of PEGA resin were compared by using fluorescently labelled peptides. A clear difference in the peptide stability towards cleavage by subtilisin, trypsin, or papain was observed. However, all of the on‐bead peptides provided an optimal surface for cell adhesion and proliferation. In long‐term experiments, these properties were still found to be similar on the resins modified either with l ‐ or with d‐ amino acids and unaffected by the nature of their fluorescence labelling at either terminus. These results support that the more accessible l ‐amino acids can be utilized for cell adhesion experiments and confirm the nonspecific interaction mechanism of cell binding to these peptides on the bead surface. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. 相似文献