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Christoph Syldatk Vera Lehmensiek Gaby Ulrichs Ulrich Bilitewski Karsten Krohn Hartmut Höke Fritz Wagner 《Biotechnology letters》1992,14(2):99-104
Summary Resting cells of a mutant ofArthrobacter sp. (DSM 3747) were used for the bioconversion of D,L-5-benzylhydantoin and related compounds to the corresponding L-amino acids. After optimization of the reaction conditions in shake flask experiments, bioconversions were performed in a preparative scale in a 2-l-bioreactor under nitrogen atmosphere. Specific productivities of 0.4 (p-NO2-L-phenylalanine) up to 3.9 mM amino acid x g cell dry mass–1 x h–1 (p-Cl-L-phenylalanine) were obtained. D,L-5-p-COOH-Benzylhydantoin, D,L-5-phenylhydantoin and D,L-5-p-OH-phenylhydantoin were not accepted as substrates. 相似文献
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Understanding the structure and function of protein complexes and multi‐domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein/protein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon S ortase‐mediated and Na tive chemical ligation using synthetic Pe ptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps – sortase‐mediated and native chemical ligation – together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
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Giant protoplasts of Saccharomyces cerevisiae of 10-35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 ± 0.07 µF/cm2) and conductance (23-44 µS/cm2) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels. 相似文献
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Debarati Paul Neha Rastogi Ulrich Krauss Michael Schlomann Gunjan Pandey Janmejay Pandey Anuradha Ghosh Rakesh K. Jain 《Indian journal of microbiology》2008,48(2):279-286
Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism
of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic
machinery of microbial degradation of several mono- phenolic and biphenolic compounds including nitrophenols. Assessment of
the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great significance from basic as well as applied study point of view. In the present study we have evaluated
the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils.
The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed
wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained
in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the
taxonomic ranks of the host microorganisms. 相似文献
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Olivia Sveidahl Johansen Tao Ma Jakob Bondo Hansen Lasse Kruse Markussen Renate Schreiber Laia Reverte-Salisa Hua Dong Dan Ploug Christensen Wenfei Sun Thorsten Gnad Iuliia Karavaeva Thomas Svava Nielsen Sander Kooijman Cheryl Cero Oksana Dmytriyeva Yachen Shen Maria Razzoli Shannon L. O’Brien Zachary Gerhart-Hines 《Cell》2021,184(13):3502-3518.e33
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Rebecca C. Schreiber Stacey A. Vaccariello Kristen Boeshore Annette M. Shadiack Richard E. Zigmond 《Developmental neurobiology》2002,53(1):68-79
Transecting the axons of neurons in the adult superior cervical ganglion (SCG; axotomy) results in the survival of most postganglionic neurons, the influx of circulating monocytes, proliferation of satellite cells, and changes in neuronal gene expression. In contrast, transecting the afferent input to the SCG (decentralization) results in nerve terminal degeneration and elicits a different pattern of gene expression. We examined the effects of decentralization on macrophages in the SCG and compared the results to those previously obtained after axotomy. Monoclonal antibodies were used to identify infiltrating (ED1+) and resident (ED2+) macrophages, as well as macrophages expressing MHC class II molecules (OX6+). Normal ganglia contained ED2+ cells and OX6+ cells, but few infiltrating macrophages. After decentralization, the number of infiltrating ED1+ cells increased in the SCG to a density about twofold greater than that previously seen after axotomy. Both the densities of ED2+ and OX6+ cells were essentially unchanged after decentralization, though a large increase in OX6+ cells occurred after axotomy. Proliferation among the ganglion's total non‐neuronal cell population was examined and found to increase about twofold after decentralization and about fourfold after axotomy. Double‐labeling experiments indicated that some of these proliferating cells were macrophages. After both surgical procedures, the percentage of proliferating ED2+ macrophages increased, while neither procedure altered the proliferation of ED1+ macrophages. Axotomy, though not decentralization, increased the proliferation of OX6+ cells. Future studies must address what role(s) infiltrating and/or resident macrophages play in regions of decentralized and axotomized neurons and, if both are involved, whether they play distinct roles. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 68–79, 2002 相似文献