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1.
Increasing markets for biopharmaceuticals, including monoclonal antibodies, have triggered a permanent need for bioprocess optimization. Biochemical engineering approaches often include the optimization of basal and feed media to improve productivities of Chinese hamster ovary (CHO) cell cultures. Often, l ‐tyrosine is added as dipeptide to deal with its poor solubility at neutral pH. Showcasing IgG1 production with CHO cells, we investigated the supplementation of three l ‐tyrosine (TYR, Y) containing dipeptides: glycyl‐l ‐tyrosine (GY), l ‐tyrosyl‐l ‐valine (YV), and l ‐prolyl‐l ‐tyrosine (PY). While GY and YV led to almost no phenotypic and metabolic differences compared to reference samples, PY significantly amplified TYR uptake thus maximizing related catabolic activity. Consequently, ATP formation was roughly four times higher upon PY application than in reference samples.  相似文献   

2.
3.
Twenty crystal structures of the complexes of l ‐asparaginase with l ‐Asn, l ‐Asp, and succinic acid that are currently available in the Protein Data Bank, as well as 11 additional structures determined in the course of this project, were analyzed in order to establish the level of conservation of the geometric parameters describing interactions between the substrates and the active site of the enzymes. We found that such stereochemical relationships are highly conserved, regardless of the organism from which the enzyme was isolated, specific crystallization conditions, or the nature of the ligands. Analysis of the geometry of the interactions, including Bürgi–Dunitz and Flippin–Lodge angles, indicated that Thr12 (Escherichia coli asparaginase II numbering) is optimally placed to be the primary nucleophile in the most likely scenario utilizing a double‐displacement mechanism, whereas catalysis through a single‐displacement mechanism appears to be the least likely.  相似文献   

4.
A pair of l ‐leucine (l ‐Leu) and d ‐leucine (d ‐Leu) was incorporated into α‐aminoisobutyric acid (Aib) peptide segments. The dominant conformations of four hexapeptides, Boc‐l ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1a), Boc‐d ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1b), Boc‐Aib‐Aib‐l ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2a), and Boc‐Aib‐Aib‐d ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2b), were investigated by IR, 1H NMR, CD spectra, and X‐ray crystallographic analysis. All peptides 1a,b and 2a,b formed 310‐helical structures in solution. X‐ray crystallographic analysis revealed that right‐handed (P) 310‐helices were present in 1a and 1b and a mixture of right‐handed (P) and left‐handed (M) 310‐helices was present in 2b in their crystalline states. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

5.
The inhibition in the synthesis or bioavailability of nitric oxide (NO) has an important role in progress of hypertension. The blocking of nitric oxide synthase activity may cause vasoconstriction with the formation of reactive oxygen species (ROS). Propolis is a resinous substance collected by honey bees from various plants. Propolis has biological and pharmacological properties. The aim of this study was to examine the effect of propolis on catalase (CAT) activity, malondialdehyde (MDA) and NO levels in the testis tissues of hypertensive rats by Nω‐nitro‐l ‐arginine methyl ester (l ‐NAME). Rats have received nitric oxide synthase inhibitor (l ‐NAME, 40 mg kg?1, intraperitoneally) for 15 days to produce hypertension and propolis (200 mg kg?1, by gavage) during the last 5 days. MDA level in l ‐NAME‐treated group significantly increased compared with control group (P < 0.01). MDA level of l ‐NAME + propolis‐treated rats significantly reduced (P < 0.01) compared with l ‐NAME‐treated group. CAT activity and NO level significantly reduced (P < 0.01) in l ‐NAME group compared with control group. There were no statistically significant increases in the CAT activity and NO level of the l ‐NAME + propolis group compared with the l ‐NAME‐treated group (P > 0.01). These results suggest that propolis changes CAT activity, NO and MDA levels in testis of l ‐NAME‐treated animals, and so it may modulate the antioxidant system. Copyright © 2013 John Wiley & Sons, Ltd.  相似文献   

6.
The mannosylated derivative of adamant‐1‐yl tripeptide (D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant‐1‐yl tripeptide (Man‐OCH2CH(Me)CO‐D ‐(Ad‐1‐yl)Gly‐L ‐Ala‐D ‐isoGln) is a non‐pyrogenic, H2O‐soluble, and non‐toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, β‐D ‐GlcNAc‐(1→4)‐D ‐MurNAc‐L ‐Ala‐D ‐isoGln‐mesoDAP(εNH2)‐D ‐Ala‐D ‐Ala), a well‐known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM.  相似文献   

7.
In the present study, we report synthesis and biological evaluation of the N‐Boc‐protected tripeptides 4a–l and N‐For protected tripeptides 5a–l as new For‐Met‐Leu‐Phe‐OMe (fMLF‐OMe) analogues. All the new ligands are characterized by the C‐terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a–l and the antagonism of 4a–l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF‐OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with ‐CH3 and ‐C(CH3)3, respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i , containing ‐F and ‐I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

8.
Melanosome movement represents a good model of cytoskeleton‐mediated transport of organelles in eukaryotic cells. We recently observed that inhibiting nitric oxide synthase (NOS) with Nω‐nitro‐l ‐arginine methyl ester (l ‐NAME) induced dispersion in melanophores pre‐aggregated with melatonin. Activation of cyclic adenosine 3′,5′‐monophosphate (cAMP)‐dependent protein kinase (PKA) or calcium‐dependent protein kinase (PKC) is known to cause dispersion. Also, PKC and NO have been shown to regulate the mitogen/extracellular signal‐regulated kinase (MEK)‐ERK pathway. Accordingly, our objective was to further characterize the signaling pathway of l ‐NAME‐induced dispersion. We found that the dispersion was decreased by staurosporine and PD98059, which respectively inhibit PKC and MEK, but not by the PKA inhibitor H89. Furthermore, Western blotting revealed that ERK1 kinase was phosphorylated in l ‐NAME‐dispersed melanophores. l ‐NAME also caused dispersion in latrunculin‐B‐treated cells, suggesting that this effect is not due to inhibition of the melatonin signaling pathway. Summarizing, we observed that PKC and MEK inhibitors decreased the l ‐NAME‐induced dispersion, which caused phosphorylation of ERK1. Our results also suggest that NO is a negative regulator of phosphorylations that leads to organelle transport.  相似文献   

9.

Aims

To increase the l ‐isoleucine production in Corynebacterium glutamicum by overexpressing the global regulator Lrp and the two‐component export system BrnFE.

Methods and Results

The brnFE operon and the lrp gene were cloned into the shuttle vector pDXW‐8 individually or in combination. The constructed plasmids were transformed into an l ‐isoleucine‐producing strain C. glutamicum JHI3‐156, and the l ‐isoleucine production in these different strains was analysed and compared. More l ‐isoleucine was produced when only Lrp was expressed than when only BrnFE was expressed. Significant increase in l ‐isoleucine production was observed when Lrp and BrnFE were expressed in combination. Compared to the control strain, l ‐isoleucine production in JHI3‐156/pDXW‐8‐lrpbrnFE increased 63% in flask cultivation, and the specific yield of l ‐isoleucine increased 72% in fed‐batch fermentation.

Conclusions

Both Lrp and BrnFE are important to enhance the l ‐isoleucine production in C. glutamicum.

Significance and Impact of the Study

The results provide useful information to enhance l ‐isoleucine or other branched‐chain amino acid production in C. glutamicum.  相似文献   

10.
Campylobacter jejuni, a major food‐borne intestinal pathogen, preferentially utilizes a few specific amino acids and some organic acids such as pyruvate and l ‐ and d ‐lactate as carbon sources, which may be important for growth in the avian and mammalian gut. Here, we identify the enzymatic basis for C. jejuni growth on l ‐lactate. Despite the presence of an annotated gene for a fermentative lactate dehydrogenase (cj1167), no evidence for lactate excretion could be obtained in C. jejuni NCTC 11168, and inactivation of the cj1167 gene did not affect growth on lactate as carbon source. Instead, l ‐lactate utilization in C. jejuni NCTC 11168 was found to proceed via two novel NAD‐independent l ‐LDHs; a non‐flavin iron–sulfur containing three subunit membrane‐associated enzyme (Cj0075c‐73c), and a flavin and iron–sulfur containing membrane‐associated oxidoreductase (Cj1585c). Both enzymes contribute to growth on l ‐lactate, as single mutants in each system grew as well as wild‐type on this substrate, while a cj0075c cj1585c double mutant showed no l ‐lactate oxidase activity and did not utilize or grow on l ‐lactate; d ‐lactate‐dependent growth was unaffected. Orthologues of Cj0075c‐73c (LldEFG/LutABC) and Cj1585c (Dld‐II) were recently shown to represent two novel families of l ‐ and d ‐lactate oxidases; this is the first report of a bacterium where both enzymes are involved in l ‐lactate utilization only. The cj0075c‐73c genes are located directly downstream of a putative lactate transporter gene (cj0076c, lctP), which was also shown to be specific for l ‐lactate. The avian and mammalian gut environment contains dense populations of obligate anaerobes that excrete lactate; our data indicate that C. jejuni is well equipped to use l ‐ and d ‐lactate as both electron‐donor and carbon source.  相似文献   

11.
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   

12.
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   

13.
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   

14.
15.
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   

16.
The influence of l ‐homoarginine on the heat‐induced aggregation of three model proteins, i.e. porcine, mink, and human growth hormones was investigated by circular dichroism spectroscopy. It was found that the effect of l ‐homoarginine as an analogue of arginine depends on the concentration of the additive as well as the protein itself. l ‐Homoarginine increased the onset temperature of heat‐induced aggregation of both porcine and mink growth hormones. However, the formation of human growth hormone aggregates was increased at low concentrations of l ‐homoarginine. Only at higher concentrations of the additive was the onset temperature of human growth hormone aggregation found to increase. Additional experiments of human growth hormone melting in the presence of histidine, lysine, and sodium chloride were performed. The effect of lysine was similar as in the presence of l ‐homoarginine. It follows that in protein formulations low concentrations of amino acids should be used with some precaution. At low concentration of additive, depending on the charge of both protein and amino acid used, the promotion of aggregation of unfolding intermediates may occur. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:808–814, 2015  相似文献   

17.
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   

18.
The nitrogen cycle initiates direct reduction of N2 to NH3 by enzymatic reactions. We hypothesize that l ‐dihydroxyphenylalanine (l ‐DOPA), a catecholamine, could be a source of nitric oxide (NO). In order to determine whether l ‐DOPA generates NO and induces any biological change in the eye, we measured the generation of NO in vitro and in vivo, and investigated the histopathological changes caused by injection of l ‐DOPA into the vitreous of rats. We also hypothesized that melanin granules may affect the generation of NO during the metabolism of l ‐DOPA, since l ‐DOPA is a precursor of melanin in the brain and the eye. Therefore, we compared the effects of l ‐DOPA on the generation of NO between amelanotic and melanotic rats. NO was measured as diffusion currents by NO electrodes. In vitro, various concentrations of l ‐DOPA (5, 29.9, 79.4, 152.7, and 249 μM) were added to the medium. The inhibition of NO generation by 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazole‐1‐oxyl 3‐oxide (carboxy‐PTIO) was tested. In vivo, NO generation in the vitreous of rats was measured and the eyes were enucleated under anesthesia after l ‐DOPA injection. The ocular tissues were subjected to histological examination. NO was produced from l ‐DOPA in a dose‐dependent manner and was scavenged by carboxy‐PTIO in vitro. NO in the vitreous of melanotic rats was generated from l ‐DOPA. Histological examination with hematoxylin‐eosin staining revealed vasodilation in the ciliary vessels and the choroid after l ‐DOPA injection. Both effects were greater in melanotic rats than in amelanotic rats. The vasodilation may be attributable to NO as well as to superoxides, which can be regulated by the existence of melanin.  相似文献   

19.
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   

20.
l ‐Cysteine is an endogenous sulfur‐containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson′s and Alzheimer′s disease. l ‐Cysteine can modulate the activity of ionic channels, including voltage‐gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l ‐cysteine on responses mediated by homomeric GABAAρ1 receptors, which are known for mediating tonic γ‐aminobutyric acid (GABA) responses in retinal neurons. GABAAρ1 receptors were expressed in Xenopus laevis oocytes and GABA‐evoked chloride currents recorded by two‐electrode voltage‐clamp in the presence or absence of l ‐cysteine. l ‐Cysteine antagonized GABAAρ1 receptor‐mediated responses; inhibition was dose‐dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration‐response curves for GABA were shifted to the right in the presence of l ‐cysteine without a substantial change in the maximal response. l ‐Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N‐ethyl maleimide. Our results suggest that redox modulation is not involved during l ‐cysteine actions and that l ‐cysteine might be acting as a competitive antagonist of the GABAAρ1 receptors.

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