全文获取类型
收费全文 | 21976篇 |
免费 | 501篇 |
国内免费 | 872篇 |
专业分类
23349篇 |
出版年
2023年 | 112篇 |
2022年 | 266篇 |
2021年 | 267篇 |
2020年 | 257篇 |
2019年 | 307篇 |
2018年 | 340篇 |
2017年 | 274篇 |
2016年 | 304篇 |
2015年 | 620篇 |
2014年 | 1678篇 |
2013年 | 1733篇 |
2012年 | 1647篇 |
2011年 | 2375篇 |
2010年 | 2042篇 |
2009年 | 1097篇 |
2008年 | 1032篇 |
2007年 | 907篇 |
2006年 | 819篇 |
2005年 | 709篇 |
2004年 | 649篇 |
2003年 | 647篇 |
2002年 | 427篇 |
2001年 | 277篇 |
2000年 | 275篇 |
1999年 | 315篇 |
1998年 | 328篇 |
1997年 | 289篇 |
1996年 | 272篇 |
1995年 | 332篇 |
1994年 | 313篇 |
1993年 | 247篇 |
1992年 | 235篇 |
1991年 | 224篇 |
1990年 | 155篇 |
1989年 | 161篇 |
1988年 | 128篇 |
1987年 | 125篇 |
1986年 | 97篇 |
1985年 | 167篇 |
1984年 | 197篇 |
1983年 | 157篇 |
1982年 | 163篇 |
1981年 | 79篇 |
1980年 | 110篇 |
1979年 | 80篇 |
1978年 | 28篇 |
1977年 | 26篇 |
1976年 | 19篇 |
1974年 | 8篇 |
1973年 | 10篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
991.
Astaxanthin preparation by fermentation of esters from Haematococcus pluvialis algal extracts with Stenotrophomonas species
下载免费PDF全文
![点击此处可从《Biotechnology progress》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Natural astaxanthin (Ax) is an additive that is widely used because of its beneficial biochemical functions. However, the methods used to produce free Ax have drawbacks. Chemical saponification methods produce several by‐products, and lipase‐catalyzed hydrolysis methods are not cost effective. In this study, a bacterial strain of Stenotrophomonas sp. was selected to enzymatically catalyze the saponification of Ax esters to produce free all‐trans‐Ax. Through single‐factor experiments and a Box–Behnken design, the optimal fermentation conditions were determined as follows: a seed culture age of 37.79 h, an inoculum concentration of 5.92%, and an initial broth pH of 6.80. Under these conditions, a fermentation curve was drawn, and the optimal fermentation time was shown to be 60 h. At 60 h, the degradation rate of the Ax esters was 98.08%, and the yield of free all‐trans‐Ax was 50.130 μg/mL. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:649–656, 2016 相似文献
992.
Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor
下载免费PDF全文
![点击此处可从《Biotechnology progress》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Stephanie A. Parker Mitchell H. Maloy Jaime Tome‐Amat Cameron L. Bardliving Carl A. Batt Kaylee J. Lanz Jonathon T. Olesberg Mark A. Arnold 《Biotechnology progress》2016,32(2):518-526
The production of norovirus virus‐like particles (NoV VLPs) displaying NY‐ESO‐1 cancer testis antigen in Pichia pastoris BG11 Mut+ has been enhanced through feed‐strategy optimization using a near‐infrared bioprocess monitor (RTBio® Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real‐time. The production of NoV VLPs displaying NY‐ESO‐1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two‐fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP‐NY‐ESO‐1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L?1 during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1‐NY‐ESO‐1 yield of 0.85 g L?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518–526, 2016 相似文献
993.
Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss
下载免费PDF全文
![点击此处可从《Biotechnology progress》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Joseph M. Newton Desmond Schofield Joanna Vlahopoulou Yuhong Zhou 《Biotechnology progress》2016,32(4):1069-1076
Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction‐point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069–1076, 2016 相似文献
994.
Selective fermentation of carbohydrate and protein fractions of Scenedesmus,and biohydrogenation of its lipid fraction for enhanced recovery of saturated fatty acids
下载免费PDF全文
![点击此处可从《Biotechnology and bioengineering》网站下载免费的PDF全文](/ch/ext_images/free.gif)
995.
Towards the industrialization of new biosurfactants: Biotechnological opportunities for the lactone esterase gene from Starmerella bombicola
下载免费PDF全文
![点击此处可从《Biotechnology and bioengineering》网站下载免费的PDF全文](/ch/ext_images/free.gif)
996.
Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances. 相似文献
997.
Jiaxin Zhang Ali Movahedi Zhiheng Wei Ming Sang Xiaolong Wu Mengyang WangHui Wei Huixin PanTongming Yin Qiang Zhuge 《Analytical biochemistry》2016
The antimicrobial peptide ABP-dHC-cecropin A is a small cationic peptide with potent activity against a wide range of bacterial species. Evidence of antifungal activity has also been suggested; however, evaluation of this peptide has been limited due to the low expression of cecropin proteins in Escherichia coli. To improve the expression level of ABP-dHC-cecropin A in E. coli, tandem repeats of the ABP-dHC-cecropin A gene were constructed and expressed as fusion proteins (SUMO-nABP-dHC-cecropin, n = 1, 2, 3, 4) via pSUMO-nABP-dHC-cecropin A vectors (n = 1, 2, 3, 4). Comparison of the expression levels of soluble SUMO-nABP-dHC-cecropin A fusion proteins (n = 1, 2, 3, 4) suggested that BL21 (DE3)/pSUMO-3ABP-dHC-cecropin A is an ideal recombinant strain for ABP-dHC-cecropin A production. Under the selected conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the expression level of ABP-dHC-cecropin A was as high as 65 mg/L, with ∼21.3% of the fusion protein in soluble form. By large-scale fermentation, protein production reached nearly 300 mg/L, which is the highest yield of ABP-dHC-cecropin A reported to date. In antibacterial experiments, the efficacy was approximately the same as that of synthetic ABP-dHC-cecropin A. This method provides a novel and effective means of producing large amounts of ABP-dHC-cecropin A. 相似文献
998.
Yasunori Sugiyama Sho Yamashita Yuuki Uezato Yukako Senga Syouichi Katayama Naoki Goshima Yasushi Shigeri Noriyuki Sueyoshi Isamu Kameshita 《Analytical biochemistry》2016
To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca2+/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities. 相似文献
999.
Sucharita Bhattacharyya Jonathan P. Renn Houqing Yu John F. Marko Andreas Matouschek 《Analytical biochemistry》2016
The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein. 相似文献
1000.
Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002
下载免费PDF全文
![点击此处可从《Biotechnology and bioengineering》网站下载免费的PDF全文](/ch/ext_images/free.gif)