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71.
E. A. Markvicheva N. E. Tkachuk S. V. Kuptsova T. N. Dugina S. M. Strukova Yu. E. Kirssh V. P. Zubov L. D. Rumish 《Applied biochemistry and biotechnology》1996,61(1-2):75-84
A new one-step procedure for entrapping proteases into a polymeric composite calcium alginate-poly(N-vinyl caproladam) hydrogel was developed that provided 75–90% retention of the activity of entrapped enzymes compared to
soluble ones. Properties of entrapped carboxypeptidase B, trypsin, and thrombin were investigated. The immobilized enzymes
were active within a wide pH range. The temperature optima of entrapped trypsin and carboxypeptidase B were approx 25°C higher
than that of the soluble enzymes, and the resistance to heating was also increased. The effects of various polar and nonpolar
organic solvents on the entrapped proteases were investigated. The immobilized enzymes retained their activity within a wide
concentration range (up to 90%) of organic solvents. Gel-entrapped trypsin and carboxypeptidase (CPB) were successfully used
for obtaining human insulin from recombinant proinsulin. The developed stabilization method can be used to catalyze various
reactions proceeding within wide pH and temperature ranges. 相似文献
72.
73.
Lionel Pochet Marc DieuRaphaël Frédérick Ann-Marie MurrayIsabelle Kempen Bernard PirotteBernard Masereel 《Tetrahedron》2003,59(25):4557-4561
6-Chloromethylcoumarin derivatives are known to express a marked inhibitory potency against serine proteases. However, their mechanism of inhibition remains unclear. In order to confirm the postulated mechanism, we use mass spectrometry. The shift mass obtained after inactivation by two compounds, which differ only by the nature of the leaving group (chloride or acetate) was in agreement with an alkylenzyme formation. With another compound devoid of a latent alkylating group, the shift mass obtained with the complex corresponds to an acylenzyme resulting from the interaction of the serine residue with the lactone carbonyl group. These results clearly demonstrate that the inhibition is not due to an attack of the exocyclic carbonyl group by the active serine but rather result from a nucleophilic attack on the intracyclic carbonyl group. 相似文献
74.
Harald Trauthwein Oliver MayUwe Dingerdissen Stefan BuchholzKarlheinz Drauz 《Tetrahedron letters》2003,44(19):3737-3739
The enantioselective enzymatic deamidation of (rac)-N-carbamoyl amino acid amides (Cbm-AA-NH2) to enantiopure (L)-N-carbamoyl amino acids (Cbm-AA-OH) is described for the first time. Via fast screening methods of biocatalysts several proteases like Chirazyme P1, Chirazyme P2 and Subtilisin were identified, which give conversions of up to 47% and >98% ee. This conversion is most productive on aliphatic and primary amino acids. 相似文献
75.
Kim Joong K. Starzak Maciej Preckshot George W. Marshall Robert Bajpai Rakesh K. 《Applied biochemistry and biotechnology》1994,(1):51-68
A mathematical model has been developed for the key reactions taking place during cheese ripening. It includes growth and
lysis of cells in the cheese matrix, cell-wall bound proteinases and intracellular peptidases that are released into cheese
upon cell lysis, and the production of peptides and amino acids from casein in cheese. The model parameters have been estimated
using published experimental data for cheddar cheese, and model simulations have been conducted to suggest effective means
of reducing ripening times of cheeses. The time required for ripening of cheeses can be significantly reduced by carefully
controlling the cell numbers at the beginning of cheese ripening and their proteinase and peptidase activities. 相似文献
76.
《Biomedical chromatography : BMC》2018,32(10)
In this study, we present hydrazide functionalized magnetic nanoparticles as a sorbent prepared by a new and facile method. Scanning electron microscope and Fourier transform infrared were used for characterizing the synthesized nanoparticles. The ability of the sorbent to extract N‐terminal serine and threonine peptides was evaluated. The peptides were modified by oxidation of the hydroxyl group in the 1,2‐amino alcohol structure before extraction. These aldehyde‐forms of peptides were specifically bonded to the hydrazide groups of the sorbent. The formed hydrazone bonds were cleaved in the presence of hydroxylamine reagent. Finally, the oximated peptides were released and quantified with a high‐performance liquid chromatography–diode array spectroscopy. The effects of experimental parameters including extraction time, elution time and elution volume on extraction efficiency were also investigated. The required time for the extraction process to reach equilibrium and elution time was only 8 h. The adsorption efficiency of the sorbent was 79 and 77% for peptides with N‐terminal serine and threonine, respectively. The sorbent showed good specificity for extracting the peptides. In addition, the extraction efficiency of the sorbent remained constant in the presence of a non‐N‐terminal serine and threonine peptide as interference. 相似文献
77.
Cucumisin (EC 3.4.21.25) isolated from prince melon fruit is a plant serine protease. Its milk-clotting activity was compared
with plant cysteine proteases such as papain (EC 3.4.22.2) and ficain (EC 3.4.22.3). Cucumisin was more stable than papain
under the condition of pH 7.1, 37‡C for 24 h. The milk-clotting activity of cucumisin was the same to that of papain and was
half value of that of ficain. 相似文献
78.
Zymography is a powerful technique to separate and identify different enzymatic activities on a standard acrylamide gel. For oxidation prone enzymes such as cysteine proteases however, the oxidizing species generated by electrolysis of the gel running buffer may result in partial or complete inactivation, thus compromising the final readout. This can be only partially remedied by subsequent treatment of the gel with reducing agents. We demonstrate the generation of reactive oxidizing species during electrophoresis and discovered that supplementation of the gel running buffer with a minimum of 5 mM cysteine prevents enzyme inactivation and allows retention of proteolytic activity as measured by zymography on model substrate N α‐benzoyl‐l ‐arginine p‐nitroanilide, without at the same time altering the mobilities of the gel proteins. 相似文献
79.
Not only α,β-dehydroamino acids are important constituents for a number of bioactive peptides in nature, but also they are important building blocks for a variety of synthetic amino acids in organic synthesis. Methods to prepare dehydroamino acids have been reported extensively in the literature; however, efficient and convenient protocols are still required. Here we have developed a convenient method to prepare dehydroalanine (ΔAla) and dehydroamino butyric acid (ΔAbu) derivatives derived from DL-serines and DL-threonines, respectively. 4-Toluenesulfonyl chloride (TsCl) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were employed in this procedure, which carried out activation of hydroxyl group and β-elimination in one pot. Because it is convenient and easy to handle, this method will attract the attention of synthetic chemists. 相似文献
80.
《Journal of separation science》2017,40(9):1960-1965
The metalloproteinase MP belongs to the serralysin family, which is involved in important functions such as nutrient acquisition and infection pathogenesis. Serralysin proteases in highly purified form are commonly used at the industrial level with several purposes. In this study, we set up an efficient and rapid purification protocol for MP using a p‐aminobenzamidine‐modified affinity chromatography. The affinity medium was synthesized by using p‐aminobenzamidine as affinity ligand immobilized via cyanuric chloride spacer to Sepharose 6B sorbent carrier. According to the adsorption analysis, the dissociation constant K d and theoretical maximum adsorption Q max of this medium were 24.2 μg/mL and 24.1 mg/g wet sorbent, respectively. The purity of MP was assessed by a high‐performance liquid chromatography on a TSK3000SW column and sodium dodecyl sulfate polyacrylamide gel electrophoresis, revealing values of 98.7 and ∼98%, respectively. The specific activity of purified MP was 95.6 U/mg, which is similar to values obtained through traditional purification protocols. In conclusion, our protocol could be easily employed for the rapid isolation of MP with high purity, and could be implemented for other serralysin family proteases. 相似文献