首页 | 本学科首页   官方微博 | 高级检索  
文章检索
  按 检索   检索词:      
出版年份:   被引次数:   他引次数: 提示:输入*表示无穷大
  收费全文   264篇
  免费   7篇
化学   149篇
力学   13篇
数学   55篇
物理学   54篇
  2021年   2篇
  2019年   4篇
  2018年   2篇
  2016年   7篇
  2015年   3篇
  2014年   5篇
  2013年   45篇
  2012年   7篇
  2011年   13篇
  2010年   13篇
  2009年   7篇
  2008年   8篇
  2007年   9篇
  2006年   14篇
  2005年   7篇
  2004年   11篇
  2003年   12篇
  2002年   15篇
  2001年   6篇
  2000年   4篇
  1999年   2篇
  1996年   9篇
  1995年   3篇
  1994年   3篇
  1993年   3篇
  1992年   2篇
  1991年   4篇
  1989年   4篇
  1988年   5篇
  1987年   2篇
  1986年   4篇
  1985年   1篇
  1984年   2篇
  1983年   2篇
  1982年   2篇
  1981年   3篇
  1980年   2篇
  1979年   2篇
  1978年   1篇
  1976年   2篇
  1975年   1篇
  1973年   2篇
  1972年   2篇
  1971年   2篇
  1970年   1篇
  1968年   2篇
  1961年   1篇
  1951年   1篇
  1928年   1篇
  1895年   2篇
排序方式: 共有271条查询结果,搜索用时 31 毫秒
81.
With increasing interest in nanoscience and nanotechnology, the fundamental underpinnings of what makes materials strong and durable are under critical investigation. Recent findings suggest that when materials are reduced in extent to nanoscopic proportions, they exhibit enhanced strength, specifically in the form of higher moduli than are measured on macroscopic objects of the same composition. Force-deformation behavior of nanostructures subjected to concentrated loads, such as with atomic force microscopy (AFM), can yield detailed information and insight about their local mechanical properties. We review and evaluate the effectiveness of deformation and indentation tests used in determining the elastic modulus of nanobeams, nanosprings, thin films, biological samples, dendrimers, and fluid droplets. Obstacles yet remain in the determination of absolute, quantitative modulus data at the nanoscale. In spite of basic limitations, recent developments in advanced nanomechanical techniques will facilitate improvement in our understanding of material strength and aging from molecules and colloids to the macroscale.  相似文献   
82.
83.
84.
Experimental solubility and sorptive dilation data are reported for carbon dioxide and ethane in a crosslinked poly(ethylene oxide) (XLPEO) rubbery copolymer. Five different temperatures (253 ≤ T(K) ≤ 308) were considered, with a maximum gas pressure of 2.09 MPa (20.6 atm). The polymer was prepared by photopolymerization of a solution containing 70 wt % poly(ethylene glycol) methyl ether acrylate (PEGMEA) and 30 wt % poly(ethylene glycol) diacrylate (PEGDA). Sorption isotherms were described by the Flory‐Huggins model. For each gas, the Flory‐Huggins interaction parameter was a decreasing function of temperature and did not show a composition dependence. Dilation and sorption data were combined to calculate the partial molar volume (PMV) of the gases in the polymer, which was an increasing function of temperature. Based on a comparison with literature data for a XLPEO homopolymer prepared from pure PEGDA over the same range of operating conditions, an effect of the network composition on both gas solubility and PMV was found. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 456–468, 2010  相似文献   
85.
86.
The crystal structure of BiPb2VO6 has been determined from powder diffraction data using a combination of direct methods and the novel approach of applying simulated annealing methods simultaneously to X-ray and neutron data; BiPb2VO6 is a polar, noncentrosymmetric, second harmonic generation active material and its crystal structure is one of the more complex to be solved ab initio from powder diffraction data.  相似文献   
87.
Bond valence sum calculations are used to investigate the crystal chemistry of the elpasolite-related oxyfluoride K3MoO3F3 in order to obtain insight into the type/s of structural distortion (away from an ideal, high symmetry, elpasolite type parent structure) responsible for a characteristic, highly structured, three-dimensional diffuse intensity distribution. The first required type of local structural distortion corresponds to large amplitude MoO3F3 octahedral rotations while the second is associated with O/F ordering and associated induced Mo ion shifts. Monte Carlo modelling is used to show how the latter when coupled with an appropriate local crystal chemical constraint can give rise to the observed structured diffuse scattering. The study is part of a wider search for diffraction evidence of oxygen/fluorine ordering in metal oxyfluoride systems.  相似文献   
88.
A bivariate Gaussian process with mean 0 and covariance
Σ(s, t, p)=Σ11(s, t)ρΣ12(s, t)ρΣ21(s, t)Σ22(s, t)
is observed in some region Ω of R′, where {Σij(s,t)} are given functions and p an unknown parameter. A test of H0: p = 0, locally equivalent to the likelihood ratio test, is given for the case when Ω consists of p points. An unbiased estimate of p is given. The case where Ω has positive (but finite) Lebesgue measure is treated by spreading the p points evenly over Ω and letting p → ∞. Two distinct cases arise, depending on whether Δ2,p, the sum of squares of the canonical correlations associated with Σ(s, t, 1) on Ω2, remains bounded. In the case of primary interest as p → ∞, Δ2,p → ∞, in which case p? converges to p and the power of the one-sided and two-sided tests of H0 tends to 1. (For example, this case occurs when Σij(s, t) ≡ Σ11(s, t).)  相似文献   
89.
α-Amylases are among the most widely used classes of enzymes in industry and considerable effort has gone into optimising their activities. Efforts to find better amylase mutants, such as through high-throughput screening, would be greatly aided by access to precise and robust active site titrating agents for quantitation of active mutants in crude cell lysates. While active site titration reagents designed for retaining β-glycosidases quantify these enzymes down to nanomolar levels, convenient titrants for α-glycosidases are not available. We designed such a reagent by incorporating a highly reactive fluorogenic leaving group onto unsaturated cyclitol ethers, which have been recently shown to act as slow substrates for retaining glycosidases that operate via a covalent ‘glycosyl’-enzyme intermediate. By appending this warhead onto the appropriate oligosaccharide, we developed efficient active site titration reagents for α-amylases that effect quantitation down to low nanomolar levels.

α-Amylases are among the most widely used classes of enzymes in industry and considerable effort has gone into optimising their activities.

Amylases are among the most common classes of enzymes employed in industrial settings, being used in detergents, bread, beer, biofuel, and many other sectors. Accordingly, α-amylases account for 25% of the world''s multi-billion dollar enzyme market.1,2 α-Amylases are endo-acting enzymes that cleave starch into malto-oligosaccharides, which are further degraded by exo-acting α-glucosidases, glucoamylases, β-amylases and α-glucan phosphorylases and lyases. They are found in CAZy GH families 13, 57, 119 and 126, with the vast majority in the large GH13 family.3 GH13 enzymes adopt a (β/α)8 fold with three highly conserved active site carboxylic acids.4–6 They employ a classical double-displacement mechanism7 in which one of the glutamic acids provides acid catalytic assistance to the leaving group departure while an aspartate attacks the anomeric centre, forming a covalent glycosyl enzyme intermediate. In a second step, water attacks the anomeric centre with base assistance from the glutamate residue (Fig. 1A and B).Open in a separate windowFig. 1Koshland mechanism of retaining β- and α-glycosidases (A & B). The same mechanism has been observed for the hydrolysis of “β”-valienols (C), and for “α”-valienols (D).Given their industrial importance, a huge amount of attention has been given to the discovery and improvement of α-amylases to attain optimal performance for particular applications. These approaches typically require high-throughput analysis of large numbers of gene products or mutants thereof.8–10 Identification of the best candidates then ideally requires high-throughput assay coupled with a method for determining the enzyme concentration in each sample. This can be a challenging task in the absence of purification, as would be the case for truly high-throughput approaches. The “gold standard” method to quantify active enzyme concentration is active site titration.11 Active site titrants react stoichiometrically with their target enzymes and release one equivalent of a quantifiable agent, which is typically either a chromophore or fluorophore. For enzymes that operate via a covalent intermediate, such as retaining glycosidases, the active site titrants are usually chromogenic or fluorogenic substrates that form this intermediate with a rate constant (kon) that is much greater than that for its hydrolysis (koff) – ideally with koff approaching zero.Our lab has previously developed active site titration reagents for several retaining β-glycosidases12,13 and neuraminidases.14,15 By replacing the substituent on the position adjacent to the anomeric centre of the sugar (the hydroxyl at C-2 for many monosaccharides) with a fluorine atom, both the formation and the hydrolysis of the glycosyl-enzyme intermediate are slowed, largely through inductive destabilisation of the transition state. Further incorporation of a reactive fluorogenic leaving group generates a reagent that, upon covalently inactivating the glycosidase, releases a stoichiometric and quantifiable amount of fluorophore. The fluorogenic response is then measured to determine the amount of active glycosidase that is present in solution.Unfortunately, this same strategy does not work for retaining α-glycosidases. In those cases, koff remains greater than kon, likely due to the inherently greater reactivity of the β-glycosyl-enzyme intermediate,16,17 and the compounds are simply substrates with low turnover numbers. By use of 2,2-dihalosugars with yet more reactive leaving groups, this problem could be solved in some cases, but their synthesis is challenging, and inactivation rates were low, or non-existent in some cases.18,19 Alternative approaches were called for.Recently, a new class of glycosidase substrates was reported in which the sugar moiety is replaced by an equivalently hydroxylated cyclohexene.20–23 Hydrolysis of these enol ethers likely occurs via an allylic cation of almost identical reactivity to that of the equivalent oxocarbenium ion. Glycosidases cleave these substrates via the classical Koshland mechanism7 (Fig. 1C and D), but considerably more slowly than their natural substrates. However, incorporation of a good leaving group will accelerate, relatively, the first step such that, in some cases, they act as mechanism-based inactivators making them candidates for development of an active site titrant for α-amylases.Since α-amylases are endo-acting enzymes that do not usually cleave monosaccharide glycosides, an ‘extended” oligosaccharide version containing a total of 2 or 3 sugar/pseudosugar moieties would be needed. Substrates longer than this would be prone to internal glycoside cleavage. Since 2-chloro-4-nitrophenyl maltotrioside (CNP-G3) functions as a substrate for most amylases, we focused on addition of a maltosyl unit to a valienol moiety containing a 6,8-difluorocoumarin (F2MU) leaving group at its “anomeric centre”. The low pKa of this coumarin, 4.7,14 results in a greater reactivity of the reagent and also ensures the coumarin will be deprotonated and thus fluorescent, upon release at neutral pH.Synthesis of partially protected alcohol 2 from gluconolactone 1via literature methods24 was followed by attachment of F2MU via a Mitsunobu reaction and subsequent removal of the protecting groups under acidic conditions, generating known pseudo-glycoside 3.23 To check this concept before we synthesized the longer version, we tested compound 3 as a titrant of a simple α-glucosidase and found that it did indeed titrate the enzyme (Fig. S5). Since elongation of this pseudosugar via classical organic synthesis would require substantial protecting group chemistry, we elected instead to employ an enzymatic coupling strategy using the GH13 cyclodextrin transglycosidase, CGTase. This enzyme can use glycosyl fluorides, such as α-maltosyl fluoride, to effect glycosyl transfer onto suitable acceptors. However, a significant competing reaction would involve self-condensation of glycosyl fluorides ultimately forming cyclodextrins. To avoid this problem, we employed a maltosyl fluoride donor (4), in which the 4′-hydroxyl had been capped with a methyl group.25,26 Incorporation of 4′-methoxy groups does not alter the reaction with α-amylases, as this site in the normal substrate is occupied by additional sugar residues. Thus CGTase-catalysed glycosylation between known glycosyl fluoride 4 and pseudo-glycoside 3, gave the pseudo-trisaccharide 5 in 64% isolated yield (Scheme 1).Open in a separate windowScheme 1Synthesis of titration reagent 5.With this reagent in hand, we proceeded to screen its ability to inactivate a small panel of α-amylases. As shown in Fig. 2, time-dependent inactivation was observed for all enzymes tested, with the most industrially relevant enzymes, Effusibacillus pohliae amylase (EPA) and Aspergillus oryzae amylase (AOA), being inactivated the fastest.Open in a separate windowFig. 2Time-dependent inactivation of a small panel of amylases, showing remaining % activity versus time. Red box with X: AOA (91 nM); blue square: EPA (66.7 nM); purple cross: PPA (500 nM); green triangle: HPA (125 nM). AOA = A. oryzae amylase; EPA = E. pohliae amylase; HPA = human pancreatic amylase; PPA = porcine pancreatic amylase.Kinetic parameters for inactivation were then determined by directly monitoring the release of F2MU by UV-Vis (Table 1). To determine kon and koff (Scheme 2), we monitored chromophore (F2MU) release by absorbance at 370 or 380 nm (dependent on the concentration of 5 in the measurements of each enzyme). After mixing 5 with each enzyme individually, a burst phase followed by a steady-state phase was observed. For each enzyme, this was then repeated with varying concentrations of 5. Initial rates of F2MU release versus concentration of 5 were fit to a Michaelis–Menten equation to provide kon. The rate constant of cyclitol release, koff, was determined by measuring rates of the steady-state region at a saturating concentration (5× Ki). We found that several amylases: Effusibacillus pohliae amylase (EPA), Aspergillus oryzae amylase (AOA), Rhizomucor pusillus amylase (RPA) and porcine pancreatic amylase (PPA), inactivated quickly (highest kon, lowest koff, and greatest kon/Ki), and are therefore ideal candidates for titration with compound 5. Human pancreatic amylase (HPA), on the other hand, while inactivating rapidly, binds the reagent relatively poorly.Open in a separate windowScheme 2Kinetic parameters for the hydrolysis of 5 by several amylases (at 25 °C for EPA, AOA, and RPA and 30 °C for human pancreatic amylase [HPA] and porcine pancreatic amylase [PPA])
Enzyme: K i (μM) k on (min−1) k on/Ki (min−1 nM−1) k off (min−1)
HPA30400.200.07n.d.
PPA2280.482.10.024
EPA45.01.7028.90.002
AOA73.00.344.60.001
RPA1600.251.60.004
Open in a separate windowConfirmation that the inactivation observed was a result of stoichiometric covalent derivatisation of the enzyme was obtained for three representative enzymes by monitoring the enzyme molecular weights before and after inactivation, by electrospray ionisation mass spectrometry. As shown in Table 2, Fig. 3 and in ESI Fig. S1, the mass of each enzyme after the reaction was increased by the expected 496 mass units relative to that of the unlabelled enzyme. Complete reaction is evident by the absence of any unlabelled enzyme peak after addition of 5 (Fig. 3 and S1). It should also be noted, that all glycoforms of AOA underwent complete reaction (Fig. 3).Open in a separate windowFig. 3MS-plot of AOA before (left side, blue) and after (right side, green) addition of 5 showing the expected increase of 496 mass units.Intact-MS results confirming the stoichiometric addition of pseudo-trisaccharide (+496 m/z) to each amylase
Enzyme:Enzyme (M + H+)Enzyme + 5 (M + H+)
HPA56 066.756 563.0
EPA75 190.675 686.0
AOA53 863.354 358.6
Open in a separate windowHaving demonstrated that these reagents function as effective time-dependent inactivators, we then evaluated their utility as active site titration agents. Initially, we tested the ability of compound 5 to titrate ∼100 nM EPA (Fig. 4A) and observed a classical burst of fluorescence followed by a steady-state turnover phase. The active enzyme concentration can be quantified from such plots by extrapolating the steady-state portion back to the y-intercept (t = 0) and fitting the burst to eqn (1), as described previously.11Burst = [E]0 × (kon/kon + koff)21Open in a separate windowFig. 4(A) Titration of EPA with 100 μM of 5. The release of F2MU was monitored fluorimetrically (λEx = 353 nm, λEm = 451 nm). The steady-state signal was extrapolated back to the y-axis to get the corresponding burst amplitude and fit to eqn (1) to give enzyme concentration. (B) Titration of a set of serial dilutions of EPA. Red small box with X: 100 nM; yellow triangle: 50 nM; green (small) square: 25 nM; blue-green cross: 12.5 nM; blue (large) square: 6.3 nM; pink star: 3.1 nM. Burst responses show a detectable response down to 3 nM. (C) Plot of dilution factor versus the calculated [Novamyl], based on the burst response in Fig. 4B with the colours corresponding to the concentrations indicated in Fig. 4B.The values we obtained in this way are shown in Table 3 and compared with the concentrations claimed by the manufacturers: agreement was excellent for EPA and AOA. To explore a case in which total protein concentration was likely to be different from active concentration, we titrated a commercial porcine pancreatic amylase (PPA) that was purchased from Sigma-Aldrich several years prior to this set of experiments. The concentration of the active enzyme was found to be almost 4-fold lower than the original stated value. This discrepancy is likely due to the degradation of the enzyme over the long storage period. Indeed, such discrepancies in active enzyme concentration and total protein concentration are exactly what the titration agent is designed to detect.Concentrations of several amylases determined by titration with reagent 5, compared with manufacturers'' listed concentrationa
Enzyme:[Manufacturers] (nM)[Titration] (nM)
AOA9193
EPA100116
PPA610164
Yeast α-glucosidaseb88007500
Open in a separate windowaDetermined by declared activity measurements.bSubstrate 3 was used for titration of this enzyme. The manufacturer''s concentration was determined by measuring the enzyme activity with PNP-Glc (Vmax) and using their reported units of activity vs. PNP-Glc.Finally, the sensitivity and linearity of the response of our titration reagent 5 were tested using a crude, commercial batch of EPA (5 mg mL−1; 66.7 μM). After initial dilution of this batch of EPA to 100 nM, a series of 2× dilutions gave a reliable and detectable fluorogenic response down to enzyme concentrations as low as 3 nM (Fig. 4B and C).  相似文献   
90.
We have studied the photochemical quantum yields of singlet oxygen production (using the RNO bleaching method) and superoxide production (using the EPR-spin trapping method and the SOD-inhibitable ferricytochrome c reduction spectral assay) of kynurenine (Ky), N-formylkynurenine (NFK), 3-hydroxykynurenine (3HK), kynurenic acid (KUA), and the flavins, riboflavin (RF) and flavin mononucleotide (FMN). Such a study of the photodynamic efficiencies is important since these compounds appear endogenously in the eye. The singlet oxygen quantum yields of the flavins and KUA are high, while Ky and 3HK generate no detectable amounts of singlet oxygen. The superoxide quantum yields of the sensitizers are low compared to their singlet oxygen, and Ky and 3HK produce no detectable amounts of superoxide. The production of the superoxide radical is enhanced in the presence of electron donor molecules such as EDTA and NADH. These results suggest that the production of oxyradicals in the lens may be modulated by the presence of endogenous electron donor molecules such as the coenzymes NADH and NADPH, which are present in significant amounts in some lenses. They also suggest that Ky and 3HK, which are known to be present in aged lenses, might play a protective rather than a deleterious role in the eye.  相似文献   
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号