全文获取类型
收费全文 | 106篇 |
免费 | 21篇 |
专业分类
127篇 |
出版年
2022年 | 2篇 |
2021年 | 2篇 |
2020年 | 1篇 |
2019年 | 1篇 |
2018年 | 1篇 |
2017年 | 4篇 |
2016年 | 1篇 |
2015年 | 9篇 |
2014年 | 4篇 |
2013年 | 16篇 |
2012年 | 8篇 |
2011年 | 3篇 |
2010年 | 5篇 |
2009年 | 3篇 |
2008年 | 6篇 |
2007年 | 4篇 |
2006年 | 4篇 |
2005年 | 5篇 |
2004年 | 6篇 |
2003年 | 1篇 |
2002年 | 2篇 |
2000年 | 2篇 |
1999年 | 2篇 |
1998年 | 2篇 |
1997年 | 2篇 |
1992年 | 4篇 |
1991年 | 3篇 |
1990年 | 1篇 |
1989年 | 1篇 |
1988年 | 1篇 |
1986年 | 1篇 |
1985年 | 1篇 |
1984年 | 1篇 |
1983年 | 2篇 |
1981年 | 2篇 |
1979年 | 2篇 |
1978年 | 3篇 |
1975年 | 1篇 |
1973年 | 2篇 |
1972年 | 3篇 |
1969年 | 1篇 |
1967年 | 1篇 |
1966年 | 1篇 |
排序方式: 共有127条查询结果,搜索用时 31 毫秒
51.
Devon L. Johnstone Michael F. O'Connell Friso P. Palstra Daniel E. Ruzzante 《Molecular ecology》2013,22(9):2394-2407
We describe temporal changes in the genetic composition of a small anadromous Atlantic salmon (Salmo salar) population from South Newfoundland, an area where salmon populations are considered threatened (COSEWIC 2010). We examined the genetic variability (13 microsatellite loci) in 869 out‐migrating smolt and post‐spawning kelt samples, collected from 1985 to 2011 for a total of 22 annual collections and a 30 year span of assigned cohorts. We estimated the annual effective number of breeders (Nb) and the generational effective population size (Ne) through genetic methods and demographically using the adult sex ratio. Comparisons between genetic and demographic estimates show that the adult spawners inadequately explain the observed Ne estimates, suggesting that mature male parr are significantly increasing Nb and Ne over the study period. Spawning as parr appears to be a viable and important strategy in the near absence of adult males. 相似文献
52.
Holwerda SW Fulton D Eubank WL Keller DM 《American journal of physiology. Heart and circulatory physiology》2011,301(4):H1639-H1645
There are important differences in autonomic function and cardiovascular responsiveness between African Americans (AA) and Caucasian Americans (CA). This study tested the hypothesis that carotid baroreflex (CBR) responsiveness is impaired in normotensive AA compared with normotensive CA at rest. CBR control of heart rate (HR) and mean arterial blood pressure (MAP) was assessed in 30 nonhypertensive male subjects (15 AA; 15 CA; age 18-33 yr) with 5-s periods of neck pressure (NP; simulated hypotension) and neck suction (NS; simulated hypertension) ranging from +45 to -80 Torr during rest. Carotid-cardiac stimulus-response curves revealed a significantly lower minimum HR response in the CA compared with AA (40.8 ± 2.4 vs. 49.8 ± 2.9 beats/min, respectively; P < 0.05). In addition, the magnitude of the mean HR response to all trials of NS (-20, -40, -60, and -80 Torr) was attenuated in the AA group (AA, -10.1 ± 1.7 vs. CA, -14.9 ± 2.2 beats/min; P < 0.05), while no significant differences were found in the magnitude of the mean HR response to NP (+15, +30, and +45 Torr) between racial groups. There were no significant differences in the carotid-vasomotor stimulus-response curves between racial groups. Also, while no racial differences were found in the magnitude of the mean MAP response to all trials of NS, the magnitude of the mean MAP response to all trials of NP was attenuated in the AA group (AA, 7.2 ± 1.3 vs. CA, 9.3 ± 1.1 mmHg; P < 0.05). Together, these findings support inherent differences in short-term blood pressure regulation between racial groups that exhibit different relative risk for the development of hypertension. 相似文献
53.
Alexandra Bezler Alexander Woglar Fabian Schneider Friso Douma Lo Bürgy Coralie Busso Pierre Gnczy 《Molecular biology of the cell》2022,33(8)
The centriole is a minute cylindrical organelle present in a wide range of eukaryotic species. Most centrioles have a signature ninefold radial symmetry of microtubules that is imparted to the axonemes of the cilia and flagella they template, with nine centriolar microtubule doublets growing into nine axonemal microtubule doublets. There are exceptions to the ninefold symmetrical arrangement of axonemal microtubules in some species, with lower or higher fold symmetries. In the few cases where this has been examined, such alterations in axonemal symmetries are grounded in similar alterations in centriolar symmetries. Here, we examine the question of microtubule number continuity between centriole and axoneme in flagellated gametes of the gregarine Lecudina tuzetae, which have been reported to exhibit a sixfold radial symmetry of axonemal microtubules. We used time-lapse differential interference microscopy to identify the stage at which flagellated gametes are present. Thereafter, using electron microscopy and ultrastructure-expansion microscopy coupled to stimulated emission depletion superresolution imaging, we uncover that a six- or fivefold radial symmetry in the axoneme is accompanied by an eightfold radial symmetry in the centriole. We conclude that the transition between centriolar and axonemal microtubules can be characterized by unexpected plasticity. 相似文献
54.
Posttranslational modifications (PTMs) of proteins greatly expand proteome diversity, increase functionality, and allow for rapid responses, all at relatively low costs for the cell. PTMs play key roles in plants through their impact on signaling, gene expression, protein stability and interactions, and enzyme kinetics. Following a brief discussion of the experimental and bioinformatics challenges of PTM identification, localization, and quantification (occupancy), a concise overview is provided of the major PTMs and their (potential) functional consequences in plants, with emphasis on plant metabolism. Classic examples that illustrate the regulation of plant metabolic enzymes and pathways by PTMs and their cross talk are summarized. Recent large-scale proteomics studies mapped many PTMs to a wide range of metabolic functions. Unraveling of the PTM code, i.e. a predictive understanding of the (combinatorial) consequences of PTMs, is needed to convert this growing wealth of data into an understanding of plant metabolic regulation.The primary amino acid sequence of proteins is defined by the translated mRNA, often followed by N- or C-terminal cleavages for preprocessing, maturation, and/or activation. Proteins can undergo further reversible or irreversible posttranslational modifications (PTMs) of specific amino acid residues. Proteins are directly responsible for the production of plant metabolites because they act as enzymes or as regulators of enzymes. Ultimately, most proteins in a plant cell can affect plant metabolism (e.g. through effects on plant gene expression, cell fate and development, structural support, transport, etc.). Many metabolic enzymes and their regulators undergo a variety of PTMs, possibly resulting in changes in oligomeric state, stabilization/degradation, and (de)activation (Huber and Hardin, 2004), and PTMs can facilitate the optimization of metabolic flux. However, the direct in vivo consequence of a PTM on a metabolic enzyme or pathway is frequently not very clear, in part because it requires measurements of input and output of the reactions, including flux through the enzyme or pathway. This Update will start out with a short overview on the major PTMs observed for each amino acid residue (PTMs, including determination of the localization within proteins (i.e. the specific residues) and occupancy. Challenges in dealing with multiple PTMs per protein and cross talk between PTMs will be briefly outlined. We then describe the major physiological PTMs observed in plants as well as PTMs that are nonenzymatically induced during sample preparation (PTMs, in particular for enzymes in primary metabolism (Calvin cycle, glycolysis, and respiration) and the C4 shuttle accommodating photosynthesis in C4 plants (PTMs observed in plants
Open in a separate window
Open in a separate window
Open in a separate windowThere are many recent reviews focusing on specific PTMs in plant biology, many of which are cited in this Update. However, the last general review on plant PTMs is from 2010 (Ytterberg and Jensen, 2010); given the enormous progress in PTM research in plants over the last 5 years, a comprehensive overview is overdue. Finally, this Update does not review allosteric regulation by metabolites or other types of metabolic feedback and flux control, even if this is extremely important in the regulation of metabolism and (de)activation of enzymes. Recent reviews for specific pathways, such as isoprenoid metabolism (Kötting et al., 2010; Banerjee and Sharkey, 2014; Rodríguez-Concepción and Boronat, 2015), tetrapyrrole metabolism (Brzezowski et al., 2015), the Calvin-Benson cycle (Michelet et al., 2013), starch metabolism (Kötting et al., 2010; Geigenberger, 2011; Tetlow and Emes, 2014), and photorespiration (Hodges et al., 2013) provide more in-depth discussions of metabolic regulation through various posttranslational mechanisms. Many of the PTMs that have been discovered in the last decade through large-scale proteomics approaches have not yet been integrated in such pathway-specific reviews, because these data are not always easily accessible and because the biological significance of many PTMs is simply not yet understood. We hope that this Update will increase the general awareness of the existence of these PTM data sets, such that their biological significance can be tested and incorporated in metabolic pathways. 相似文献
Amino Acid Residue | Observed Physiological PTM in Plants | PTMs Caused by Sample Preparation |
---|---|---|
Ala (A) | Not known | |
Arg (R) | Methylation, carbonylation | |
Asn (N) | Deamidation, N-linked gycosylation | Deamidation |
Asp (D) | Phosphorylation (in two-component system) | |
Cys (C) | Glutathionylation (SSG), disulfide bonded (S-S), sulfenylation (-SOH), sulfonylation (-SO3H), acylation, lipidation, acetylation, nitrosylation (SNO), methylation, palmitoylation, phosphorylation (rare) | Propionamide |
Glu (E) | Carboxylation, methylation | Pyro-Glu |
Gln (Q) | Deamidation | Deamidation, pyro-Glu |
Gly (G) | N-Myristoylation (N-terminal Gly residue) | |
His (H) | Phosphorylation (infrequent) | Oxidation |
Ile (I) | Not known | |
Leu (L) | Not known | |
Lys (K) | N-ε-Acetylation, methylation, hydroxylation, ubiquitination, sumoylation, deamination, O-glycosylation, carbamylation, carbonylation, formylation | |
Met (M) | (De)formylation, excision (NME), (reversible) oxidation, sulfonation (-SO2), sulfoxation (-SO) | Oxidation, 2-oxidation, formylation, carbamylation |
Phe (F) | Not known | |
Pro (P) | Carbonylation | Oxidation |
Ser (S) | Phosphorylation, O-linked glycosylation, O-linked GlcNAc (O-GlcNAc) | Formylation |
Thr (T) | Phosphorylation, O-linked glycosylation, O-linked GlcNAc (O-GlcNAc), carbonylation | Formylation |
Trp (W) | Glycosylation (C-mannosylation) | Oxidation |
Tyr (Y) | Phosphorylation, nitration | |
Val (V) | Not known | |
Free NH2 of protein N termini | Preprotein processing, Met excision, formylation, pyro-Glu, N-myristoylation, N-acylation (i.e. palmitoylation), N-terminal α-amine acetylation, ubiquitination | Formylation (Met), pyro-Glu (Gln) |
Table II.
Most significant and/or frequent PTMs observed in plantsType of PTM (Reversible, Except if Marked with an Asterisk) | Spontaneous (S; Nonenzymatic) or Enzymatic (E) | Comment on Subcellular Location and Frequency |
---|---|---|
Phosphorylation (Ser, Thr, Tyr, His, Asp) | E | His and Asp phosphorylation have low frequency |
S-Nitrosylation (Cys) and nitration* (Tyr) | S (RNS), but reversal is enzymatic for Cys by thioredoxins | Throughout the cell |
Acetylation (N-terminal α-amine, Lys ε-amine) | E | In mitochondria, very little N-terminal acetylation, but high Lys acetylation; Lys acetylation correlates to [acetyl-CoA] |
Deamidation (Gln, Asn) | S, but reversal of isoAsp is enzymatic by isoAsp methyltransferase | Throughout the cell |
Lipidation (S-acetylation, N-meristoylation*, prenylation*; Cys, Gly, Lys, Trp, N terminal) | E | Not (or rarely) within plastids, mitochondria, peroxisomes |
N-Linked glycosylation (Asp); O linked (Lys, Ser, Thr, Trp) | E | Only proteins passing through the secretory system; O linked in the cell wall |
Ubiquination (Lys, N terminal) | E | Not within plastids, mitochondria, peroxisomes |
Sumoylation (Lys) | E | Not within plastids, mitochondria, peroxisomes |
Carbonylation* (Pro, Lys, Arg, Thr) | S (ROS) | High levels in mitochondria and chloroplast |
Methylation (Arg, Lys, N terminal) | E | Histones (nucleus) and chloroplasts; still underexplored |
Glutathionylation (Cys) | E | High levels in chloroplasts |
Oxidation (Met, Cys) | S (ROS) and E (by PCOs; see Fig. 1B), but reversal is enzymatic by Met sulfoxide reductases, glutaredoxins, and thioredoxins, except if double oxidized | High levels in mitochondria and chloroplast |
Peptidase* (cleavage peptidyl bond) | E | Throughout the cell |
S-Guanylation (Cys) | S (RNS) | Rare; 8-nitro-cGMP is signaling molecule in guard cells |
Formylation (Met) | S, but deformylation is enzymatic by peptide deformylase | All chloroplasts and mitochondria-encoded proteins are synthesized with initiating formylated Met |
Table III.
Regulation by PTMs in plant metabolism and classic examples of well-studied enzymes and pathwaysMany of these enzymes also undergo allosteric regulation through cellular metabolites. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; PRK, phosphoribulokinase.Process | Enzymes | PTMs, Protein Modifiers, Localization | References |
---|---|---|---|
Calvin-Benson cycle (chloroplasts) | Many enzymes | Oxidoreduction of S-S bonds, reversible nitrosylation, glutathionylation; through ferredoxin/ferredoxin-thioredoxin reductase/thioredoxins (mostly f and m) and glutaredoxins; proteomics studies in Arabidopsis and C. reinhardtii | Michelet et al. (2013) |
Rubisco | Methylation, carbamylation, acetylation, N-terminal processing, oligomerization; classical studies in pea (Pisum sativum), spinach (Spinacia oleracea), and Arabidopsis | Houtz and Portis (2003); Houtz et al. (2008) | |
GAPDH/CP12/PRK supercomplex | Dynamic heterooligomerization through reversible S-S bond formation controlled by thioredoxins | Graciet et al. (2004); Michelet et al. (2013); López-Calcagno et al. (2014) | |
Glycolysis | Cytosolic PEPC | Phosphorylation (S, T), monoubiquitination | O’Leary et al. (2011) |
Photorespiration | Seven enzymes are phosphorylated | Phosphorylation from meta-analysis of public phosphoproteomics data for Arabidopsis; located in chloroplasts, peroxisomes, mitochondria | Hodges et al. (2013) |
Maize glycerate kinase | Redox-regulated S-S bond; thioredoxin f; studied extensively in chloroplasts of C4 maize | Bartsch et al. (2010) | |
Respiration (mitochondria) | Potentially many enzymes, but functional/biochemical consequences are relatively unexplored | Recent studies suggested PTMs for many tricarboxylic acid cycle enzymes, including Lys acetylation and thioredoxin-driven S-S formation; in particular, succinate dehydrogenase and fumarase are inactivated by thioredoxins | Lázaro et al. (2013); Schmidtmann et al. (2014); Daloso et al. (2015) |
PDH | Ser (de)phosphorylation by intrinsic kinase and phosphatase; ammonia and pyruvate control PDH kinase activity; see Figure 1B | Thelen et al. (2000); Tovar-Méndez et al. (2003) | |
C4 cycle (C3 and C4 homologs also involved in glycolysis and/or gluconeogenesis) | Pyruvate orthophosphate dikinase | Phosphorylation by pyruvate orthophosphate dikinase-RP, an S/T bifunctional kinase-phosphatase; in chloroplasts | Chastain et al. (2011); Chen et al. (2014) |
PEPC | Phosphorylation; allosteric regulation by malate and Glc-6-P; in cytosol in mesophyll cells in C4 species (e.g. Panicum maximum); see Figure 1A | Izui et al. (2004); Bailey et al. (2007) | |
PEPC kinase | Ubiquitination resulting in degradation (note also diurnal mRNA levels and linkage to activity level; very low protein level); in cytosol in mesophyll cells in C4 species (e.g. Flaveria spp. and maize) | Agetsuma et al. (2005) | |
PEPC kinase | Phosphorylation in cytosol in bundle sheath cells | Bailey et al. (2007) | |
Starch metabolism (chloroplasts) | ADP-Glc pyrophosphorylase | Redox-regulated disulfide bonds and dynamic oligomerization; thioredoxins; see Figure 1C | Geigenberger et al. (2005); Geigenberger (2011) |
Starch-branching enzyme II | Phosphorylation by Ca2+-dependent protein kinase; P-driven heterooligomerization | Grimaud et al. (2008); Tetlow and Emes (2014) | |
Suc metabolism (cytosol) | SPS (synthesis of Suc) | (De)phosphorylation; SPS kinase and SPS phosphatase; 14-3-3 proteins; cytosol (maize and others) | Huber (2007) |
Suc synthase (breakdown of Suc) | Phosphorylation; Ca2+-dependent protein kinase; correlations to activity, localization, and turnover | Duncan and Huber (2007); Fedosejevs et al. (2014) | |
Photosynthetic electron transport (chloroplast thylakoid membranes) | PSII core and light-harvesting complex proteins | (De)phosphorylation by state-transition kinases (STN7/8) and PP2C phosphatases (PBCP and PPH1/TAP38) | Pesaresi et al. (2011); Tikkanen et al. (2012); Rochaix (2014) |
Nitrogen assimilation | Nitrate reductase | (De)phosphorylation; 14-3-3 proteins | Lillo et al. (2004); Huber (2007) |
55.
Michaël C. Fontaine Kathleen Roland Isabelle Calves Frederic Austerlitz Friso P. Palstra Krystal A. Tolley Sean Ryan Marisa Ferreira Thierry Jauniaux Angela Llavona Bayram Öztürk Ayaka A. Öztürk Vincent Ridoux Emer Rogan Marina Sequeira Ursula Siebert Gísli A. Vikingsson Asunción Borrell Johan R. Michaux Alex Aguilar 《Molecular ecology》2014,23(13):3306-3321
Despite no obvious barriers to gene flow in the marine realm, environmental variation and ecological specializations can lead to genetic differentiation in highly mobile predators. Here, we investigated the genetic structure of the harbour porpoise over the entire species distribution range in western Palearctic waters. Combined analyses of 10 microsatellite loci and a 5085 base‐pair portion of the mitochondrial genome revealed the existence of three ecotypes, equally divergent at the mitochondrial genome, distributed in the Black Sea (BS), the European continental shelf waters, and a previously overlooked ecotype in the upwelling zones of Iberia and Mauritania. Historical demographic inferences using approximate Bayesian computation (ABC) suggest that these ecotypes diverged during the last glacial maximum (c. 23–19 kilo‐years ago, kyrbp ). ABC supports the hypothesis that the BS and upwelling ecotypes share a more recent common ancestor (c. 14 kyrbp ) than either does with the European continental shelf ecotype (c. 28 kyrbp ), suggesting they probably descended from the extinct populations that once inhabited the Mediterranean during the glacial and post‐glacial period. We showed that the two Atlantic ecotypes established a narrow admixture zone in the Bay of Biscay during the last millennium, with highly asymmetric gene flow. This study highlights the impacts that climate change may have on the distribution and speciation process in pelagic predators and shows that allopatric divergence can occur in these highly mobile species and be a source of genetic diversity. 相似文献
56.
57.
Martinelli N Trabetti E Pinotti M Olivieri O Sandri M Friso S Pizzolo F Bozzini C Caruso PP Cavallari U Cheng S Pignatti PF Bernardi F Corrocher R Girelli D 《PloS one》2008,3(2):e1523
Background
Relative little attention has been devoted until now to the combined effects of gene polymorphisms of the hemostatic pathway as risk factors for Myocardial Infarction (MI), the main thrombotic complication of Coronary Artery Disease (CAD). The aim of this study was to evaluate the combined effect of ten common prothrombotic polymorphisms as a determinant of MI.Methodology/Principal Findings
We studied a total of 804 subjects, 489 of whom with angiographically proven severe CAD, with or without MI (n = 307; n = 182; respectively). An additive model considering ten common polymorphisms [Prothrombin 20210G>A, PAI-1 4G/5G, Fibrinogen β -455G>A, FV Leiden and “R2”, FVII -402G>A and -323 del/ins, Platelet ADP Receptor P2Y12 -744T>C, Platelet Glycoproteins Ia (873G>A), and IIIa (1565T>C)] was tested. The prevalence of MI increased linearly with an increasing number of unfavorable alleles (χ2 for trend = 10.68; P = 0.001). In a multiple logistic regression model, the number of unfavorable alleles remained significantly associated with MI after adjustment for classical risk factors. As compared to subjects with 3-7 alleles, those with few (≤2) alleles had a decreased MI risk (OR 0.34, 95%CIs 0.13–0.93), while those with more (≥8) alleles had an increased MI risk (OR 2.49, 95%CIs 1.03–6.01). The number of procoagulant alleles correlated directly (r = 0.49, P = 0.006) with endogenous thrombin potential.Conclusions
The combination of prothrombotic polymorphisms may help to predict MI in patients with advanced CAD. 相似文献58.
Alissa M. Williams Giulia Friso Klaas J. van Wijk Daniel B. Sloan 《The Plant journal : for cell and molecular biology》2019,98(2):243-259
Eukaryotic cells represent an intricate collaboration between multiple genomes, even down to the level of multi‐subunit complexes in mitochondria and plastids. One such complex in plants is the caseinolytic protease (Clp), which plays an essential role in plastid protein turnover. The proteolytic core of Clp comprises subunits from one plastid‐encoded gene ( clpP1 ) and multiple nuclear genes. The clpP1 gene is highly conserved across most green plants, but it is by far the fastest evolving plastid‐encoded gene in some angiosperms. To better understand these extreme and mysterious patterns of divergence, we investigated the history of clpP1 molecular evolution across green plants by extracting sequences from 988 published plastid genomes. We find that clpP1 has undergone remarkably frequent bouts of accelerated sequence evolution and architectural changes (e.g. a loss of introns and RNA ‐editing sites) within seed plants. Although clpP1 is often assumed to be a pseudogene in such cases, multiple lines of evidence suggest that this is rarely true. We applied comparative native gel electrophoresis of chloroplast protein complexes followed by protein mass spectrometry in two species within the angiosperm genus Silene , which has highly elevated and heterogeneous rates of clpP1 evolution. We confirmed that clpP1 is expressed as a stable protein and forms oligomeric complexes with the nuclear‐encoded Clp subunits, even in one of the most divergent Silene species. Additionally, there is a tight correlation between amino acid substitution rates in clpP1 and the nuclear‐encoded Clp subunits across a broad sampling of angiosperms, suggesting continuing selection on interactions within this complex. 相似文献
59.
Friso R. Postma Trudi Hengeveld Jacqueline Alblas Ben N.G. Giepmans Gerben C.M. Zondag Kees Jalink Wouter H. Moolenaar 《The Journal of cell biology》1998,140(5):1199-1209
Gap junctions mediate cell–cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell–cell communication is rapidly disrupted by G protein–coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1–2 h of receptor stimulation. In contrast, a desensitization-defective G protein–coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein–coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell–cell communication. 相似文献
60.
Sonke T Ernste S Tandler RF Kaptein B Peeters WP van Assema FB Wubbolts MG Schoemaker HE 《Applied and environmental microbiology》2005,71(12):7961-7973
An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60 degrees C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+ (to 80%), Mn2+ (to 400%), and Mg2+ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of alpha-hydrogen- and (bulky) alpha,alpha-disubstituted alpha-amino acid amides, alpha-hydroxy acid amides, and alpha-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, beta-amino and beta-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry. 相似文献