全文获取类型
收费全文 | 135篇 |
免费 | 9篇 |
出版年
2023年 | 1篇 |
2021年 | 2篇 |
2020年 | 1篇 |
2018年 | 1篇 |
2016年 | 1篇 |
2015年 | 5篇 |
2014年 | 6篇 |
2013年 | 5篇 |
2012年 | 6篇 |
2011年 | 6篇 |
2010年 | 3篇 |
2009年 | 6篇 |
2008年 | 7篇 |
2007年 | 5篇 |
2006年 | 9篇 |
2005年 | 4篇 |
2004年 | 5篇 |
2003年 | 8篇 |
2002年 | 6篇 |
2001年 | 9篇 |
2000年 | 5篇 |
1999年 | 5篇 |
1993年 | 2篇 |
1992年 | 4篇 |
1991年 | 3篇 |
1990年 | 2篇 |
1989年 | 2篇 |
1988年 | 2篇 |
1987年 | 4篇 |
1986年 | 3篇 |
1985年 | 4篇 |
1984年 | 3篇 |
1982年 | 1篇 |
1981年 | 2篇 |
1980年 | 1篇 |
1979年 | 3篇 |
1978年 | 1篇 |
1977年 | 1篇 |
排序方式: 共有144条查询结果,搜索用时 31 毫秒
21.
Inouye K Minoda M Takita T Sakurama H Hashida Y Kusano M Yasukawa K 《Protein expression and purification》2006,46(2):248-255
Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin. 相似文献
22.
Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile. 相似文献
23.
Sekiguchi S Hashida Y Yasukawa K Inouye K 《Bioscience, biotechnology, and biochemistry》2012,76(1):95-100
Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay. In this study, we evaluated the effects of sugars on the kinetic stability of BIALP in the hydrolysis of p-nitrophenylphosphate (pNPP). The temperatures reducing initial activity by 50% in a 30-min incubation, T(50), of BIALP with 1.0 M disaccharide (sucrose and trehalose) or 2.0 M monosaccharide (glucose and fructose) were 55.0-55.5 °C, 4.7-5.2 °C higher than without sugar (50.3±0.1 °C). The T(50) of BIALP increased to 58.4±0.3 °C when the trehalose concentration was from 1.0 to 1.5 M, but did not change when the glucose concentration was from 2.0 to 3.0 M. Thermodynamic analysis revealed that the stabilization of BIALP by sugars was driven by the increase in the enthalpy change of activation for thermal inactivation of BIALP. No sugars affected the k(cat) of BIALP in the hydrolysis of pNPP. These results suggest that not only trehalose, which is considered the most effective stabilizer of enzymes, but also sucrose, glucose, and fructose can be used as stabilizers of BIALP. 相似文献
24.
Mari L. Shinohara Michiko Ihara Masanobu Abo Miyoko Hashida Shinobu Takagi Thomas C. Beck 《Applied microbiology and biotechnology》2001,57(5-6):653-659
A branching enzyme (EC 2.4.1.18) gene was isolated from an extremely thermophilic bacterium, Rhodothermus obamensis. The predicted protein encodes a polypeptide of 621 amino acids with a predicted molecular mass of 72 kDa. The deduced amino acid sequence shares 42-50% similarity to known bacterial branching enzyme sequences. Similar to the Bacillus branching enzymes, the predicted protein has a shorter N-terminal amino acid extension than that of the Escherichia coli branching enzyme. The deduced amino acid sequence does not appear to contain a signal sequence, suggesting that it is an intracellular enzyme. The R. obamensis branching enzyme was successfully expressed both in E. coli and a filamentous fungus, Aspergillus oryzae. The enzyme showed optimum catalytic activity at pH 6.0-6.5 and 65 degrees C. The enzyme was stable after 30 min at 80 degrees C and retained 50% of activity at 80 degrees C after 16 h. Branching activity of the enzyme was higher toward amylose than toward amylopectin. This is the first thermostable branching enzyme isolated from an extreme thermophile. 相似文献
25.
26.
27.
Nomizu K Hashida K Makino R Ohara S 《Bioscience, biotechnology, and biochemistry》2008,72(7):1682-1689
The most efficient steaming conditions below 200 degrees C for extracting antioxidants from used tea leaves and their reaction behavior during the steaming treatment were investigated. The antioxidative activity of the steamed extracts increased with increasing steaming temperature, and the yield of the ethyl acetate extract fraction from each steamed extract showing the greatest antioxidative activity also increased. Caffeine, (-)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, (-)-catechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, (-)-epigallocatechin gallate and gallic acid were identified from the ethyl acetate extract fraction. Quantitative analyses demonstrated that the catechins with a 2,3-cis configuration decreased with increasing steaming temperature, whereas the corresponding epimers at the C-2 position increased. Each pair of epimers showed similar antioxidative activity to each other, indicating that the epimerization reaction did not contribute to the improved antioxidative activity. It is concluded from these results that the improvement in antioxidative activity at higher steaming temperatures was due to the increased yield of catechins and other antioxidants. 相似文献
28.
Nakai Y Hashida H Kadota K Minami M Shimizu K Matsumoto I Kato H Abe K 《Bioscience, biotechnology, and biochemistry》2008,72(1):139-148
The functional balance between brown adipose tissue (BAT) and white adipose tissue (WAT) is important for metabolic homeostasis. We compared the effects of fasting on the gene expression profiles in BAT, WAT and liver by using a DNA microarray analysis. Tissues were obtained from rats that had been fed or fasted for 24 h. Taking the false discovery rate into account, we extracted the top 1,000 genes that had been differentially expressed between the fed and fasted rats. In all three tissues, a Gene Ontology analysis revealed that the lipid and protein biosynthesis-related genes had been markedly down-regulated. The whole-body fuel shift from glucose to triacylglycerol and the induction of autophagy were also observed. There was marked up-regulation of genes in the 'protein ubiquitination' category particularly in BAT of the fasted rats, suggesting that the ubiquitin-proteasome system was involved in saving energy as an adaptation to food shortage. 相似文献
29.