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181.
Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants 总被引:3,自引:1,他引:2
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Martin Dominik Vollmer Helga Hoier Hans-Jürgen Hecht Ursula Schell Janosch Grning Adrian Goldman Michael Schlmann 《Applied microbiology》1998,64(9):3290-3299
Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other. 相似文献
182.
Influence of morphology and rheology on the production characteristics of the basidiomycete Cyathus striatus 总被引:3,自引:0,他引:3
The influence of the pellet morphology of the basidiomycete Cyathus striatus on the production of the antibiotics striatals A, B, and C was investigated. The main operating parameters in fermenters of different sizes were the tip speed and the volumetric power input. Different methods were developed for quantification of morphological characteristics. The apparent viscosity of the suspension was measured with a cylinder rheometer. Sediment density was measured with a sedimentation apparatus. Particle size distributions were recorded with an image analysis system. By means of the presented measuring methods, morphological characteristics could be determined and enabled an early assessment of the fermentation course and the antibiotics production. During the exponential growth phase of the fungus the relative sediment height correlated with the biomass concentration. The pellet morphology at this stage influenced the later production of striatals. The yield of the striatals was markedly influenced by pellet size and sediment density. Since these morphological characteristics determine the rheological properties of the culture the measurement of the apparent viscosity of the culture in the production phase allowed predictions of the production yield. 相似文献
183.
Xanthomonas campestris pv. campestris gum Mutants: Effects on Xanthan Biosynthesis and Plant Virulence 总被引:4,自引:0,他引:4
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Federico Katzen Diego U. Ferreiro Cristian G. Oddo M. Vernica Ielmini Anke Becker Alfred Pühler Luis Ielpi 《Journal of bacteriology》1998,180(7):1607-1617
Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490–2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non-gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant. 相似文献
184.
Christian Schobert Lucian Baker Judit Szederkényi Pia Großmann Ewald Komor Hiroaki Hayashi Mitsuo Chino William J. Lucas 《Planta》1998,206(2):245-252
The mature, functional sieve-tube system in higher plants is dependent upon protein import from the companion cells to maintain
a functional long-distance transport system. Soluble proteins present within the sieve-tube lumen were investigated by analysis
of sieve-tube exudates which revealed the presence of distinct sets of polypeptides in seven monocotyledonous and dicotyledonous
plant species. Antibodies directed against sieve-tube exudate proteins from Ricinus communis L. demonstrated the presence of shared antigens in the phloem sap collected from Triticum aestivum L., Oryza sativa L., Yucca filamentosa L., Cucurbita maxima Duch., Robinia pseudoacacia L. and Tilia platyphyllos L. Specific antibodies were employed to identify major polypeptides. Molecular chaperones related to Rubisco-subunit-binding
protein and cyclophilin, as well as ubiquitin and the redox proteins, thioredoxin h and glutaredoxin, were detected in the
sieve-tube exudate of all species examined. Actin and profilin, a modulator of actin polymerization, were also present in
all analyzed phloem exudates. However, some proteins were highly species-specific, e.g. cystatin, a protease-inhibitor was
present in R. communis but was not detected in exudates from other species, and orthologs of the well-known squash phloem lectin, phloem protein
2, were only identified in the sieve-tube exudate of R. communis and R. pseudoacacia. These findings are discussed in terms of the likely roles played by phloem proteins in the maintenance and function of the
enucleate sieve-tube system of higher plants.
Received: 12 February 1998 / Accepted: 16 March 1998 相似文献
185.
大鼠催乳素基因真核细胞可表达性质粒的构建及应用研究 总被引:4,自引:0,他引:4
735bp的PRLcDNA片段从质粒PRL-SP65#1中回收后,用粘性末端连接法将其重组到真核表达载体pcDNA3上,筛选出正向连接重组体pcDNA3-PRLS和反向连接重组体pcDNA3-PRLAS。将重组体pcDNA3-PRLs和空载体pcDNA3分别转入NIH3T3细胞系,用G418筛选出阳性细胞后与未转染的NIH3T3细胞在加E2和不加E2的情况下,用原位杂交的方法,分别用PRLcDNA探针和原癌基因c-H-rascDNA探针进行检测,未转染的NIH3T3细胞在加E2和不加E2时都几乎无催乳素基因的表达,同样,转入空载体的NIH3T3细胞也无PRL的表达,而转入重组体pcDNA3-PRLS的NIH3T3细胞则有大量的PRL基因的表达,与对照组相比有显著差异(P<0.01)。正常和转入空载体的NIH3T3细胞有一定程度的原癌基因c-H-ras的表达,当分别加入E2和转入重组体pcDNA3-PRLS后,NIH3T3细胞中的c-H-ras基因表达水平都显著升高(P<0.05)。 相似文献
186.
Christian van den Bos Anke Rammes Thomas Vogl Raymond Boynton Joseph Zaia Clemens Sorg Johannes Roth 《Protein expression and purification》1998,13(3):313-318
A method is described for purification of P6, MRP8, and MRP14, three calcium-binding proteins assigned to the S100 protein family. The purification procedure included preparation of human granulocytes, ammonium sulfate precipitation, and anion-exchange chromatography and resulted in the copurification of P6, MRP8, and MRP14. Individual proteins were separated by either preparative isoelectric focusing or preparative SDS–PAGE. The procedure was carried out in the course of 4 days and yielded several milligrams of essentially pure P6, MRP8, and MRP14 in either native or denatured form. 相似文献
187.
β-Glucosidase from almonds (EC 3.2.1.21) was covalently immobilized by a two-step technique. In the first step, double bonds
were introduced into the β-glucosidase by derivatization with itaconic anhydride. In separate studies with α-N-protected l-amino acids, it was established that itaconic anhydride acylated mainly primary amino groups of lysines and, to a much lesser
extent hydroxyl groups of tyrosines and sulfhydryl groups of cysteines. The acylated β-glucosidase showed no loss of activity
and the K
m decreased from 3.6 mM to 2.6 mM when p-nitrophenyl β-d-glucopyranoside was used as the substrate. In the second step, the derivatized β-glucosidase was co-polymerized radically
with N,N′-methylenebisacrylamide in buffer solution. The resulting acrylamide immobilizate possessed a much better storage stability
at 30–56 °C when compared to β-glucosidase immobilized on Eupergit C. However, the specific activity was higher with the Eupergit
immobilizate. Free and acrylamide-immobilized β-glucosidase were used for glucosylation of chloramphenicol by transglucosylation
in 20% (v/v) acetonitrile at 37 °C. The acrylamide immobilizate demonstrated a great enhancement of stability and approximately
50% more chloramphenicol β-glucoside was obtained after 5 h.
Received: 22 September 1997 / Accepted: 28 October 1997 相似文献
188.
E. J. Bormann M. Leißner M. Roth B. Beer K. Metzner 《Applied microbiology and biotechnology》1998,50(5):604-607
Polyhydroxybutyrate (PHB) was produced by Ralstonia eutropha DSM 11348 (formerly Alicaligenes eutrophus) in media containing 20–30 g l−1 casein peptone or casamino acids as sole sources of nitrogen. In fermentations using media based on casein peptone, permanent
growth up to a cell dry mass of 65 g l−1 was observed. PHB accumulated in cells up to 60%–80% of dry weight. The lowest yields were found in media without any trace
elements or with casamino acids added only. The residual cell dry masses were limited to 10–15 g l−1 and did not contain PHB. The highest productivity amounted to 1.2 g PHB l−1 h−1. The mean molecular mass of the biopolymer was determined as 750 kDa. The proportion of polyhydroxyvalerate was less than
0.2% in PHB. The bioprocess was scaled up to a 300-l plant. During a fermentation time of 39 h the cells accumulated PHB to
78% w/w. The productivity was 0.98 g PHB l−1 h1.
Received: 8 July 1998 / Accepted: 26 August 1998 相似文献
189.
190.
Bjoern von Einem Anke Wahler Tobias Schips Alberto Serrano-Pozo Christian Proepper Tobias M. Boeckers Angelika Rueck Thomas Wirth Bradley T. Hyman Karin M. Danzer Dietmar R. Thal Christine A. F. von Arnim 《PloS one》2015,10(6)
Proteolytic processing of amyloid-β precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) is the initial step in the production of amyloid beta (Aβ), which accumulates in senile plaques in Alzheimer’s disease (AD). Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA) proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα), sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain. 相似文献