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介体型乙醇生物传感器的研制及应用 总被引:1,自引:0,他引:1
本文报道了电流型乙醇生物传感器的研制与应用。该传感器以健合型耐尔蓝修饰浸蜡石墨电极为基体电极,将乙醇脱氢酶及NAD^+固定在人造丝网上,成为一种无试剂的乙醇生物传感器。在pH8.8的Tris/HCl介质中,该传感器的响应电流与乙醇浓度在0.10-1.0mmol/l范围内有良好的线性关系。 相似文献
24.
Fumihiko Hasuni Katsunori Fukuoka Shuichi Adachi Yasumitsu Miyamoto Ichiro Okura 《Applied biochemistry and biotechnology》1996,56(3):341-344
Alanine synthesis by reductive amination of pyruvate was performed by the combination of NADH regeneration system and alanine
dehydrogenase (AlaDH). The conversion of pyruvate to alanine was 99% after 1 h. Leucine synthesis was also carried out by
the combination of NADH regeneration system and leucine dehydrogenase (LeuDH). The conversion of 4-methyl-2-oxovalerate to
leucine was 60% after 1.5 h. 相似文献
25.
An automated analysis system is described for the measurement of hydrogen peroxide based on a chemiluminescence reaction with phenyl 10-methylacridinium-9-carboxylate (PMAC). A reversed FIA experimental arrangement is used to establish the operating conditions for the measurement of submicromolar levels of hydrogen peroxide. The carrier stream consists of hydrogen peroxide standards prepared in a pH 9.0, boric acid buffer and the flow rate for this carrier/sample stream is 4 ml/min. Twenty microliters of a 10 mM PMAC solution, prepared in a pH 3 phosphate buffer, are injected into the carrier/sample stream. Hydrogen peroxide mixes with the PMAC reagent in an incubation coil that is constructed by wrapping 107 cm of polyethylene tubing around a 1 cm o.d. plastic rod. The chemiluminescence reaction is then initiated by adding base just before the sample passes in front of a photomultiplier tube (PMT) detector. The calculated limit of detection (S/N = 3) for hydrogen peroxide is 0.25 M. In addition, the pH dependent hydrolysis of the PMAC reagent is characterized by an HPLC method which has been specifically developed for the separation and detection of the hydrolysis products of PMAC. Results indicate that a pH of 3.0 is required for long term stability of the PMAC reagent. Finally, this system has been successfully extended to the measurement of glutamate by coupling a bioreactor column of glutamate oxidase with the hydrogen peroxide detection scheme. A detection limit (S/N = 3) of 0.5 M has been established for glutamate with a throughput of 200 samples per hour. 相似文献
26.
含离子液体[bmim]Cl的反应介质中马肝醇脱氢酶的催化特性 总被引:2,自引:0,他引:2
研究了马肝醇脱氢酶(HLADH)在含离子液体1-丁基-3-甲基咪唑氯酸盐([bmim]Cl)的反应介质中的催化特性. 以乙醇为底物时,该酶在[bmim]Cl含量≤0.15 g/ml的体系中的活力高于在不含离子液体的体系中的活力; 离子液体浓度过高(>0.15 g/ml)对酶活性有明显的抑制作用. 反应温度和pH对含离子液体的反应介质中酶活力的影响规律与不含离子液体时的规律相似. 与不含离子液体的反应介质相比, HLADH在含0.05 g/ml [bmim]Cl的体系中催化乙醇氧化的活化能下降,酶反应的Vmax和Km均升高. 反应体系中低浓度(≤0.1 g/ml)的离子液体能提高酶的热稳定性,但高浓度(>0.1 g/ml)的离子液体可降低酶的热稳定性. 紫外二阶导数光谱显示,在含不同浓度离子液体的反应介质中酶分子构象的变化有较大的差异. 相似文献
27.
Francisco M. Gírio Fátima Pelica M. T. Amaral-Collaço 《Applied biochemistry and biotechnology》1996,56(1):79-87
The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells ofDebaryomyces hansenii was partially purified in two Chromatographic steps, and characterization studies were carried out in order to inves tigate
the role of the xylitol dehydrogenase-catalyzed step in the regu lation of D-xylose metabolism. The enzyme was most active
at pH 9.0–9.5, and exhibited a broad polyol specificity. The Michaelis con stants for xylitol and NAD+ were 16.5 and 0.55 mM, respectively. Ca2+, Mg2+, and Mn2+ did not affect the enzyme activity. Conversely, Zn2+, Cd2+, and Co2+ strongly inhibited the enzyme activity. It was concluded that NAD+-xylitol dehydrogenase from D.hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and Km value for xylitol, and therefore should be named L-iditol:NAD+-5-oxidoreductase (EC 1.1.1.14). The reason D.hansenii is a good xylitol producer is not because of its value of Km for xylitol, which is low enough to assure its fast oxidation by NAD+ xylitol dehydrogenase. However, a higher Km value of xylitol dehydro genase for NAD+ compared to theK
m
values of other xylose-ferment ing yeasts may be responsible for the higher xylitol yields. 相似文献
28.
Howell EE Shukla U Hicks SN Smiley RD Kuhn LA Zavodszky MI 《Journal of computer-aided molecular design》2001,15(11):1035-1052
R67 dihydrofolate reductase (DHFR) is a novel enzyme that confers resistance to the antibiotic trimethoprim. The crystal structure of R67 DHFR displays a toroidal structure with a central active-site pore. This homotetrameric protein exhibits 222 symmetry, with only a few residues from each chain contributing to the active site, so related sites must be used to bind both substrate (dihydrofolate) and cofactor (NADPH) in the productive R67 DHFR?NADPH?dihydrofolate complex. Whereas the site of folate binding has been partially resolved crystallographically, an interesting question remains: how can the highly symmetrical active site also bind and orient NADPH for catalysis? To model this ternary complex, we employed DOCK and SLIDE, two methods for docking flexible ligands into proteins using quite different algorithms. The bound pteridine ring of folate (Fol I) from the crystal structure of R67 DHFR was used as the basis for docking the nicotinamide-ribose-Pi (NMN) moiety of NADPH. NMN was positioned by both DOCK and SLIDE on the opposite side of the pore from Fol I, where it interacts with Fol I at the pore's center. Numerous residues serve dual roles in binding. For example, Gln 67 from both the B and D subunits has several contacts with the pteridine ring, while the same residue from the A and C subunits has several contacts with the nicotinamide ring. The residues involved in dual roles are generally amphipathic, allowing them to make both hydrophobic and hydrophilic contacts with the ligands. The result is a `hot spot' binding surface allowing the same residues to co-optimize the binding of two ligands, and orient them for catalysis. 相似文献
29.
An effective method was developed to use an enzyme in ionic liquids; the asymmetric reduction of ketones by Geotrichum candidum in ionic liquids proceeded smoothly with excellent enantioselectivity when the cell was immobilized on water-absorbing polymer containing water, while the reaction without the polymer did not proceed. 相似文献
30.
Ivanauskas F. Kaunietis I. Laurinavičius V. Razumienė J. Šimkus R. 《Journal of mathematical chemistry》2005,38(3):355-366
The plate-gap model of porous enzyme doped electrode has been proposed and analyzed. It was suggested that reaction diffusion conditions in pores of bulk electrode resemble particular conditions in thin gap between parallel conducting plates. The model is based on the diffusion equations containing a nonlinear term related to the Michaelis–Menten kinetic of the enzymatic reaction inside gap. Steady state current was calculated for the wide range of given parameters and substrate concentrations. All dependences of current on substrate concentration were approximated by hyperbolas in order to obtain “apparent” parameters (maximal currents and apparent Michaelis constants) of modelled biosensors. Simple approximate relationships between given and apparent parameters were derived. The applicability of theoretical plate-gap model was tested for the case of carbon paste electrodes which were doped with PQQ – dependent glucose dehydrogenase. It was found, that soluble glucose dehydrogenase based biosensors exhibit characteristic features of the theoretical plate-gap biosensors. 相似文献