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151.
Cloning,sequence analysis,and expression of a gene encoding <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. DS-1 cholesterol oxidase 总被引:1,自引:1,他引:0
Noriyuki Doukyu Kanpei Shibata Hiroyasu Ogino Martin Sagermann 《Applied microbiology and biotechnology》2009,82(3):479-490
Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence
of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase.
The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid
residues. The amino acid sequence of the product showed significant similarity (53–62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an
active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1.
K
m and V
max values of the cholesterol oxidase were estimated from Lineweaver–Burk plots. The V
max/K
m ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral
analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures. 相似文献
152.
Saito Y Fukuhara A Nishio K Hayakawa M Ogawa Y Sakamoto H Fujii K Yoshida Y Niki E 《The Journal of nutritional biochemistry》2009,20(5):350-357
Coenzyme Q (CoQ) is a well-known electron transporter in the mitochondrial respiratory chain. Furthermore, ubiquinol (UQH(2))--a reduced form of ubiquinone (UQ)--has been shown to act as a radical-scavenging antioxidant. Some studies have reported the beneficial effect of CoQ addition to cultured cells; however, the cellular uptake and distribution of CoQ have not been elucidated. In the present study, we used rat pheochromocytoma PC12 cells to investigate and compare the cellular uptake and distribution of CoQ(10) and alpha-tocopherol (alphaT). UQ(10) or UQ(10)H(2) treatment resulted in an increase in the cellular content of both CoQ(10) in a time- and concentration-dependent manner. A subcellular fractionation study revealed that the added UQ(10) as well as UQ(10)H(2) mainly localized in the mitochondrial fraction, which is similar to the localization of endogenous CoQ but different from that of alphaT. The cellular distribution of alphaT directly corresponded to the lipid distribution, while the CoQ distribution did not show any relationship with the lipid distribution, particularly in the mitochondrial and microsomal fractions. These results indicate that the cellular distribution of CoQ is completely different from that of alphaT; moreover, a certain system which accumulates CoQ preferentially in mitochondria may be suggested. 相似文献
153.
Yusuke Miyachi Nobuaki Shimizu Chiaki Ogino Hideki Fukuda Akihiko Kondo 《Bioorganic & medicinal chemistry letters》2009,19(13):3619-3622
DNA aptamers, which bind specific molecule, such as 8-OHdG, with high affinity were investigated using an in vitro selection strategy called systematic evolution of ligands by exponential enrichment (SELEX). However, 8-OHdG was difficult to immobilize on a carrier for SELEX. Therefore, a DNA aptamer binding to 8-OHdG was selected using GMP-agarose as an analogue from a library of about 460 random ssDNA sources. As a result, three aptamer candidates were selected. Among the selected DNA aptamers, the No. 22 DNA aptamer exhibited a high affinity for 8-OHdG. The dissociation constant, KD, of No. 22 DNA aptamer was on the order of 0.1 μmol/L. This result suggests that using an analogue will be a useful new SELEX method for obtaining various aptamers that are difficult to immobilize on a matrix. 相似文献
154.
Ken Horii Takashi Adachi Takanori Tanino Tsutomu Tanaka Hiroshi Sahara Seiji Shibasaki Chiaki Ogino Yoji Hata Mitsuyoshi Ueda Akihiko Kondo 《Biotechnology letters》2009,31(8):1259-1264
A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and
molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF),
which included pepsin at pH 2. The displayed β-glucosidase resisted pepsin digestion compared with secreted, free β-glucosidase.
In SDS-PAGE and Western blotting analysis, the secreted β-glucosidase was immediately digested within 1 min following SGF
treatment, although the displayed β-glucosidase was stable for more than 60 min following SGF treatment. In addition, the
residual activity of secreted β-glucosidase was completely destroyed after 10 min SGF treatment. However, displayed β-glucosidase
retained 14% of its residual activity following the same treatment. These results clearly show that cell surface display technology
using enzymes can reveal the protease resistance of a protein of interest under various conditions. 相似文献
155.
Ken Horii Takashi Adachi Takanori Tanino Tsutomu Tanaka Atsushi Kotaka Hiroshi Sahara Tsuneharu Hashimoto Nobuyuki Kuratani Seiji Shibasaki Chiaki Ogino Hideo Noda Yoji Hata Mitsuyoshi Ueda Akihiko Kondo 《Enzyme and microbial technology》2010,46(3-4):194-199
We used cutinase from the filamentous fungi Aspergillus oryzae to produce dairy flavors. Secretory and displayed forms of cutinase were investigated using salt-free butter, which is composed mostly of triacylglycerides, as the substrate. The secretory form of cutinase, which was produced in recombinant A. oryzae, was suitable for producing butyric acids (16.8 mol%). Also, cutinase displayed on the cell surface of the yeast Saccharomyces cerevisiae as a fusion protein with α-agglutinin released butyric acid at a 2.7-fold rate (45.4 mol%) higher than that of the secreted form. Yeasts carrying two copies of cutinase genes into their chromosomes, which were constructed using the HELOH method, released free fatty acids rapidly and showed 2-fold higher lipase activity compared with yeasts carrying one copy of the cutinase gene. 相似文献
156.
Ryosuke Yamada Tsutomu Tanaka Chiaki Ogino Akihiko Kondo 《Applied microbiology and biotechnology》2010,88(4):849-857
Yeast, such as Saccharomyces cerevisiae or Kluyveromyces lactis is appropriate strain for ethanol production or some useful compounds production. Cellulases expressing yeast can ferment
ethanol from cellulosic materials; however, the productivity should be increase more and more. To improve and engineer the
productivity, the target gene(s) were introduced into yeast genome. Generally, using genetic engineering, increasing integrated
gene numbers are increased, the expressed protein ability such as enzymatic activities are also increased. In this mini-review,
we focused on the effect of integrated gene copy number and the polyploidy on the productivity such as enzymatic activity
and/or product yield. 相似文献
157.
Biotechnological production of enantiomeric pure lactic acid from renewable resources: recent achievements, perspectives, and limits 总被引:1,自引:0,他引:1
Kenji Okano Tsutomu Tanaka Chiaki Ogino Hideki Fukuda Akihiko Kondo 《Applied microbiology and biotechnology》2010,85(3):413-423
Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA
is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation; however, most
microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA production using
several kinds of genetically modified microorganisms, such as LA bacteria, Escherichia coli, Corynebacterium glutamicum, and yeast. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass has
been significantly improved. In this review, the drawbacks as well as improvements of LA production by fermentation is discussed. 相似文献
158.
Ryosuke Yamada Naho Taniguchi Tsutomu Tanaka Chiaki Ogino Hideki Fukuda Akihiko Kondo 《Microbial cell factories》2010,9(1):32
Background
The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration. 相似文献159.
Mirela Belu Michelle Javier Katayoun Ayasoufi Sarah Frischmann Catherine Jin Kuo-Chen Wang Rui Sousa-Neves Claudia Mieko Mizutani 《Journal of visualized experiments : JoVE》2010,(43)
Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos. 相似文献
160.
Tomoharu Osada Hideki Ogino Toshiaki Hino Sachiyo Ichinose Kenji Nakamura Akira Omori Toshiaki Noce Mitsuko Masutani 《PloS one》2010,5(9)