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311.
Addition of substrate-binding domains increases substrate-binding capacity and specific activity of a chitinase from Trichoderma harzianum 总被引:3,自引:0,他引:3
Chitinase Chit42 from Trichoderma harzianum CECT 2413 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin-binding domain (ChBD). We have produced hybrid chitinases with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and the cellulose-binding domain from cellobiohydrolase II of Trichoderma reesei. The chimeric chitinases had similar activities towards soluble substrate but higher hydrolytic activity than the native chitinase on high molecular mass insoluble substrates such as ground chitin or chitin-rich fungal cell walls. 相似文献
312.
313.
Takao Arimori Noriko Kawamoto Shoko Shinya Nobuo Okazaki Masami Nakazawa Kazutaka Miyatake Tamo Fukamizo Mitsuhiro Ueda Taro Tamada 《The Journal of biological chemistry》2013,288(26):18696-18706
Chitinase C from Ralstonia sp. A-471 (Ra-ChiC) has a catalytic domain sequence similar to goose-type (G-type) lysozymes and, unlike other chitinases, belongs to glycohydrolase (GH) family 23. Using NMR spectroscopy, however, Ra-ChiC was found to interact only with the chitin dimer but not with the peptidoglycan fragment. Here we report the crystal structures of wild-type, E141Q, and E162Q of the catalytic domain of Ra-ChiC with or without chitin oligosaccharides. Ra-ChiC has a substrate-binding site including a tunnel-shaped cavity, which determines the substrate specificity. Mutation analyses based on this structural information indicated that a highly conserved Glu-141 acts as a catalytic acid, and that Asp-226 located at the roof of the tunnel activates a water molecule as a catalytic base. The unique arrangement of the catalytic residues makes a clear contrast to the other GH23 members and also to inverting GH19 chitinases. 相似文献
314.
目的:实现棘孢木霉(Trichoderma asperellum)几丁质酶基因tachi2的原核高效表达,研究几丁质酶Tachi2的酶学性质.方法:利用PCR技术扩增得到几丁质酶基因tachi2,将其克隆到原核表达载体pEHISTEV中,测序后,转化大肠杆菌BL21感受态细胞,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导后进行Tachi2蛋白的纯化和复性.用纯化的目的蛋白Tachi2进行几丁质酶酶学性质的研究.结果:tachi2基因在重组大肠杆菌中正确表达,其主要以包涵体形式存在;重组蛋白Tachi2分子量约为44kDa,经过纯化和复性后得到的Tachi2有较高的几丁质酶活性.该酶的最适温度为40℃,最适pH值为7.0,几丁质酶在40℃以下比较稳定、pH 6~9时酶有较高活性,受Cu2和Zn2+的强烈抑制.结论:成功实现了棘孢木霉几丁质酶基因tachi2的原核高效表达,表达纯化了重组蛋白,明确了几丁质酶Tachi2的酶学性质,为该几丁质酶的进一步开发利用和深入研究奠定了基础. 相似文献
315.
Jogi Madhuprakash Avinash Singh Sanjit Kumar Mau Sinha Punit Kaur Sujata Sharma Appa R Podile Tej P Singh 《International Journal of Biochemistry and Molecular Biology》2013,4(4):166-178
Chitinases are known to hydrolyze chitin polymers into smaller chitooligosaccharides. Chitinase from bacterium Serratia proteamaculans (SpChiD) is found to exhibit both hydrolysis and transglycosylation activities. SpChiD belongs to family 18 of glycosyl hydrolases (GH-18). The recombinant SpChiD was crystallized and its three-dimensional structure was determined at 1.49 Å resolution. The structure was refined to an R-factor of 16.2%. SpChiD consists of 406 amino acid residues. The polypeptide chain of SpChiD adopts a (β/α)8 triosephosphate isomerase (TIM) barrel structure. SpChiD contains three acidic residues, Asp149, Asp151 and Glu153 as part of its catalytic scheme. While both Asp149 and Glu153 adopt single conformations, Asp151 is observed in two conformations. The substrate binding cleft is partially obstructed by a protruding loop, Asn30 - Asp42 causing a considerable reduction in the number of available subsites in the substrate binding site. The positioning of loop, Asn30 - Asp42 appears to be responsible for the transglycosylation activity. The structure determination indicated the presence of sulfone Met89 (SMet89). The sulfone methionine residue is located on the surface of the protein at a site where extra domain is attached in other chitinases. This is the first structure of a single domain chitinase with hydrolytic and transglycosylation activities. 相似文献
316.
A chitinase (VpChiA) and its C-terminal truncated G589 mutant (VpChiAG589) of Vibrio parahaemolyticus were cloned by polymerase chain reaction (PCR) techniques. To study the role of the C-terminal 30 amino acids of VpChiA in
the enzymatic hydrolysis of chitin, both the recombinant VpChiA and VpChiAG589 encoded in 1,881 and 1,791 bp DNA fragments,
respectively, were expressed in Escherichia coli using the pET-20b(+) expression system. The His–Tag affinity purified VpChiA and VpChiAG589 enzymes had a calculated molecular
mass of 65,713 and 62,723 Da, respectively. The results of biochemical characterization including kinetic parameters, spectroscopy
of fluorescence and circular dichroism, chitin-binding and hydrolysis, and thermostability, both VpChiA and VpChiAG589, had
very similar physicochemical properties such as the optimum pH (6), temperature (40°C), and kinetic parameters of Km and kcat against the 4MU–(GlcNAc)2 or 4MU–(GlcNAc)3 soluble substrates. The significant increase of thermostability and the drastic decrease of the hydrolyzing ability of VpChiAG589
toward the insoluble α-chitin substrate suggested that a new role could be played by the C-terminal 30 amino acids. 相似文献
317.
Maruthasalam S Kalpana K Kumar KK Loganathan M Poovannan K Raja JA Kokiladevi E Samiyappan R Sudhakar D Balasubramanian P 《Plant cell reports》2007,26(6):791-804
Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial
blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization,
and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny
analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein
in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight.
S. Maruthasalam and K. Kalpana have contributed to this article equally. 相似文献
318.
Elevated serum levels of a secreted glycoprotein chitinase 3 like 1 (CHI3L1) are associated with poor prognosis and short survival time of patients with cervical cancer (CxCa). Our previous microarray data showed the increased expression of CHI3L1 in invasive CxCa compared to normal tissue, implicating a potential role of CHI3L1 in CxCa. To establish the pathological role of CHI3L1 in the development of CxCa, this study focused on its expression in CxCa and angiogenic impacts in tumor vessel formation. CHI3L1 activated angiogenesis by promoting endothelial cell migration and tube formation in vitro but failed to protect CxCa cell lines, CaSki and HeLa against apoptosis induced by γ-irradiation. In addition, the capability of CHI3L1 to induce proliferation and migration of CaSki and HeLa cells was cell type specific. In an analysis of 103 specimens from CxCa patients, increased expression levels of CHI3L1 mRNA and protein in invasive CxCa were 4-fold (P < 0.05) and 2-fold (P < 0.01), respectively, stronger than those in normal subjects. The immunostaining of CHI3L1 was positively correlated with VEGF expression (P = 0.0019) and microvessel density (P = 0.0110). Moreover, CHI3L1 expression was also positively associated with cancer metastasis (P = 0.011). The data suggest the crucial role of CHI3L1 by promoting angiogenesis, which may contribute to the development and progression of CxCa. The findings help establish CHI3L1 as a prognostic biomarker and therapeutic target for CxCa patients. 相似文献
319.
几丁质酶是昆虫病原真菌金龟子绿僵菌致病力的主要因子之一。本实验用RT-PCR方法,从本实验室分离筛选到的高毒力金龟子绿僵菌Metarhizium anisopliae HN1中,扩增得到几丁质酶基因全长,此基因全长为1275bp,登录号为DQ011865,经Blastn分析此基因序列与M. anisopliae E6的chi1基因(AF02749)同源率为96% 。以pET-22b(+)为基础载体,构建pET-chi重组表达载体,在大肠杆菌(Escherichia. coli )BL 21中进行表达。经SDS-PAGE分析,获得了42kDa大小的重组目的蛋白,目的蛋白占表达总蛋白含量的63.3%。菌体经冷冻与超声波破碎后,按DNS法可测得几丁质酶的活性。 相似文献
320.
《Saudi Journal of Biological Sciences》2017,24(1):192-199
A halotolerant actinobacterial strain isolated from salinity affected soil of Eastern Indo-Gangetic plains (IGP), Uttar Pradesh, India, was characterised for its antagonistic potential against Macrophomina phaseolina by dual-culture assay. It was shown to effectively inhibit the growth of M. phaseolina with an inhibition zone of 27 ± 1.33 mm. Further the actinobacterial strain was evaluated for its plant growth promoting (PGP) properties and its ability to produce biocontrol related extracellular enzymes viz. amylase, protease, cellulase, chitinase, gelatinase and urease. The results revealed that the actinobacterial strain had PGP potential along with positive assay for amylase, chitinase and urease. The interaction study between antagonist strain and fungal pathogen, performed by scanning electron microscopy technique revealed that the actinobacterium was able to damage fungal mycelia may be due to chitinase, establishing its role as a potential antagonist against M. phaseolina. The actinobacterial isolate was characterised by 16S rDNA gene sequencing, and was identified as Streptomyces genera. The identified gene sequence was deposited to NCBI GenBank with an accession number KP331758. 相似文献