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101.
The synthesis of four β-lactam antibiotics (penicillin G, pivaloyloxymethyl ester of penicillin G, ampicillin and pivampicillin) catalyzed byEscherichia coli penicillin acylase has been investigated in water-methanol mixtures. The enzyme reactions were either thermodynamically or kinetically controlled at the same conditions using phenylacetic acid andd--phenylglycine methyl ester as acyl donors and 6-aminopenicillanic acid and pivaloyloxymethyl 6-aminopenicillanic acid as acyl acceptors. It has been found that the influences of the cosolvent content on the reaction rates and synthetic yields are significantly different depending on the substrates used in the experiments. On the other hand, within certain ranges of the methanol content (up to ca. 40% (v/v) the residual activities of the enzymes in water-methanol mixtures were only slightly lower than those in aqueous media. To analyze the factors that determine the reaction rate in water-cosolvent mixtures, the effect of methanol on the apparent pK values of the substrates has been investigated, and a mathematical model has been developed on the basis of the assumption that the enzyme binds non-ionized substrates. Model simulation results indicate that the solvent effect on reaction rates is mainly attributed to the kinetic effects of changes in apparent pK values. 相似文献
102.
Hee Wook Ryu Kyung Suk Cho Young Keun Chang Ho Nam Chang 《Biotechnology Techniques》1996,10(12):899-904
Summary
Alcaligenes eutrophus was successfully recovered from high cell density broths by pre-treatment with polyaluminium hydroxide chloride silicate as a coagulant at 36–90 mg Al/l. The optimum pH range for cell coagulation was 10–12. Subsequent centrifugation (45×g) and filtration (pore size 0.5 mm) gave a cell recovery of higher than 90%. The energy demand for cell recovery with the coagulant was only 3–11% of that without it. 相似文献
103.
Summary The direct, lipase-catalyzed esterifications of glycerol-3-phosphate in an organic solvent system and in a solvent free system were carried out. In a solvent free system only, LPA synthesis could be achieved within the acceptable reaction time. Open reaction system was preferable to closed reaction system for LPA synthesis. Yield of LPA isolated by silica gel column chromatography was 32.3%. 相似文献
104.
The impact of salinity on CH4 emission was studied by adding salt to a Philippine rice paddy, increasing pore water EC to approx. 4 dS.m-1 Methane emission from the salt-amended plot and adjacent control plots was monitored with a closed chamber technique. The
addition of salt to the rice field caused a reduction by 25% in CH4 emission. Rates of methane emissions from intact soil cores were measured during aerobic and anaerobic incubations. The anaerobic
CH4 fluxes from the salt-amended soil cores were three to four times lower than from cores of the control plot, whereas the aerobic
CH4 fluxes were about equal. Measurements of the potential CH4 production with depth showed that the CH4 production in the salt-amended field was strongly reduced compared to the control field. Calculation of the percentage CH4 oxidized of the anaerobic flux indicated that CH4 oxidation in the salt-amended plot was even more inhibited than CH4 production. The net result was about equal aerobic CH4 fluxes from both salt-amended plots and non-amended plots. The data illustrate the importance of both CH4 production and CH4 oxidation when estimating CH4 emission and show that the ratio between CH4 production and CH4 oxidation may depend on environmental conditions. The reduction in CH4 emission from rice paddies upon amendment with salt low in sulfate is considerably smaller than the reduction in CH4 emission observed in a similar study where fields were amended with high-sulfate containing salt (gypsum). The results indicate
that CH4 emissions from wetland rice fields on saline, low-sulfate soils are lower than CH4 emissions from otherwise comparable non-saline rice tields. However, the reduction in CH4 emission is not proportional to the reduction in CH4 production 相似文献
105.
Impaired interleukin-3 (IL-3) response of the A/J mouse is caused by a branch point deletion in the IL-3 receptor alpha subunit gene. 总被引:2,自引:0,他引:2 下载免费PDF全文
Interleukin-3 (IL-3) alone does not support hematopoietic colony formation of bone marrow cells from the A/J mouse. To elucidate the molecular lesion in A/J mice, we examined expression of the alpha and beta subunits of the IL-3 receptor (IL-3R). While IL-3R beta was normally expressed, IL-3R alpha was not detectable on the surface of A/J-derived cells by antibody staining. Genetic linkage analysis using recombinant inbred mouse strains between A/J and IL-3-responsive C57BL/6 indicated that the IL-3R alpha gene locus was responsible for the impaired IL-3 response in A/J mice. Molecular cloning and characterization of A/J-derived IL-3R alpha cDNA revealed that it lacked the sequence corresponding to exon 8, which encodes 10 amino acid residues in the extracellular domain. The aberrant splicing was due to a 5 base pair deletion at the branch point in intron 7 and was reproduced in heterologous cells by transfecting with an IL-3R alpha minigene carrying the deleterious intron. The A/J-specific abnormal form of IL-3R alpha was localized inside the cells, but not on the cell surface, providing the molecular basis for the impaired IL-3 response in the A/J mouse. 相似文献
106.
107.
Operational patterns affecting lactic acid production in ultrafiltration cell recycle bioreactor 总被引:2,自引:0,他引:2
Lactic acid production with cell recycling on an ultrafiltration tubular membrane reactor was studied; higher lactic acid concentrations as well as productivities were obtained under long-term fermentations compared with other high cell density systems. Different operational conditions, namely dilution rates and start-up modes, were assessed. Performances were very different at the three different dilution rates tested (D = 0.20 h(-1), D = 0.40 h(-1), or D = 0.58 h(-1)). The different behaviours are discussed and factors responsible for them are presented. The best way to operate for lactic acid production is chosen, the dilution rate of D = 0.40 h(-1) being the one providing the best overall performance. On the other hand, results show that of the two start-up modes tested, continuous start (membrane open) permits higher permeabilities throughout the operational runs than batch start (membrane closed). Operational stability was found to be directly associated with membranes that work at "steady state," the membrane permeability being kept around 15 L/m(2) h. Optimized cell bleed can improve time of operation if such membrane permeability can be maintained for a longer time. A comparison of results with those obtained in other lactic acid production systems is presented; such comparison shows that this tubular ultrafiltration membrane cell recycle reactor presents three important advantages: (1) concomitant lactic acid concentrations and productivities; (2) long periods of operation at reasonable permeabilities; and (3) good mechanical stability permitting the use of steam sterilization. (c) 1995 John Wiley & Sons, Inc. 相似文献
108.
Hyung-Joo Jin Jeong–Ha Kim Chul Hyun Sohn R.E. DeWreede Tae–Joo Choi G.H.N. Towers J.B. Hudson Yong–Ki Hong 《Journal of applied phycology》1997,9(4):383-388
Fifty-nine species of marine macrophytes from the coasts of British Columbia, Canada and Korea have been screened for the
presence of PCR inhibitors, namely inhibitors of Taq DNA polymerase. Eleven of the species displayed some inhibitor activity.
At the concentration of 5 μg of methanol extract in 25μL reaction mixture of PCR containing 1.5 unit of Taq DNA polymerase,
one (Ulva sp.) of 8 Chlorophyta, eight (Colpomenia bullosa, Ecklonia cava, Endarachne binghamiae, Fucus distichus, Hizikia
fusiformis, Sargassum confusum, Sargassum sagamianum, and Sargassum thunbergii) of 28 Phaeophyta, and one (Symphyocladia latiuscula)
of 34 Rhodophyta showed inhibition in PCR amplification. In the case of the water extract, two (Cladophora columbiana, Ulva
sp.) Chlorophyta, seven (Endarachne binghamiae, Fucus distichus, Hizikia fusiformis, Sargassum confusum, Sargassum sagamianum,
Sargassum horneri, Scytosiphon dotyi) Phaeophyta, no Rhodophyta and one (Phyllospadix scouleri) seagrass showed inhibition
in PCR amplification. the methanol fraction of Sargassum confusum and the water fraction of Fucus gardneri (mid–intertidal)
have been found to inhibit PCR at level as low as 0.5 μg in 25μL of PCR reaction mixture.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
109.
Mismatch Repair by Efficient Nick-Directed, and Less Efficient Mismatch-Specific, Mechanisms in Homologous Recombination Intermediates in Chinese Hamster Ovary Cells 总被引:5,自引:0,他引:5 下载免费PDF全文
Repair of single-base mismatches formed in recombination intermediates in vivo was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks (DSBs) introduced into regions of shared homology in pairs of plasmid substrates heteroallelic at 11 phenotypically silent mutations. Recombination was expected to occur primarily by single-strand annealing, yielding predicted heteroduplex DNA (hDNA) regions with three to nine mismatches. Product spectra were consistent with hDNA only occurring between DSBs. Nicks were predicted on opposite strands flanking hDNA at positions corresponding to original DSB sites. Most products had continuous marker patterns, and observed conversion gradients closely matched predicted gradients for repair initiated at nicks, consistent with an efficient nick-directed, excision-based mismatch repair system. Discontinuous patterns, seen in ~10% of products, and deviations from predicted gradients provided evidence for less efficient mismatch-specific repair, including G-A -> G-C specific repair that may reflect processing by a homologue of Escherichia coli MutY. Mismatch repair was >80% efficient, which is higher than seen previously with covalently closed, artificial hDNA substrates. Products were found in which all mismatches were repaired in a single tract initiated from one or the other nick. We also observed products resulting from two tracts of intermediate length initiated from two nicks. 相似文献
110.
Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B. 下载免费PDF全文
In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate. 相似文献