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Functional Analysis of Trehalose Synthase in Meiothermus ruber CBS-01 by Site-directed Mutation
利用定点突变分析海藻糖合酶的功能

WANG Yu-Fan,ZHU Yue-Ming,WEI Dong-Sheng,ZHANG Jun,XING Lai-Jun,LI Ming-Chun,
王宇凡
,朱玥明,魏东盛,张 峻,邢来君,李明春

微生物学通报 , 2009,
Abstract: After constructed a 3D-Model and make the multiple sequence alignment of amino acid sequences of trehalose synthase from Meiothermus ruber CBS-01, we performed site-directed mutagenesis of D200G/H165R, R227C, R392A. And the ablity of convertion was detected. D200G/H165R and R392A lost their activities basically, while the ability of convertion of R227C declined at 50°C. When reacted at 37°C, D200G/H165R lost its activity, while R392A and R227C dropped their ability. At last, we found that R392 and D200 had important role on activity of enzyme, while R227 had little affection.
红色亚栖热菌tps/tpp海藻糖合成途径中相关基因的克隆、表达及功能鉴定  [PDF]
朱玥明?,唐亦辰?,徐恒毅?,张娟?,魏东盛?,邢来君?,李明春?
生物工程学报 , 2009,
Abstract: 通过构建红色亚栖热菌(meiothermusrubercbs-01)的基因组dna文库,克隆得到该嗜热菌海藻糖合成途径中的磷酸海藻糖合成酶(tps)和磷酸海藻糖磷酸酯酶(tpp)基因。以pet21a为表达载体,将磷酸海藻糖合成酶和磷酸海藻糖磷酸酯酶在大肠杆菌中进行表达并纯化,利用薄层层析的方法验证了这两个酶的活性。同时,本研究检测了红色亚栖热菌在各种环境压力下细胞内含物成分的变化情况,发现在高渗环境压力的诱导下,该菌会在胞内积累大量的6-磷酸海藻糖,而并非海藻糖,这为进一步研究tps/tpp和tres途径在细胞体内的作用奠定了基础。
The sectionalized DNA shuffling: an effective tool for molecular directed evolution of Meiothermus ruber TreS
分段DNA shuffling: 一种大分子海藻糖合酶有效的定向进化方法

Liu Yan-Chao,Wang Yu-Fan,Qian Ke-Fan,Zhang Jun,Xiao Chen-Peng,Xing Lai-Jun,Li Ming-Chun,
刘艳超
,王宇凡,钱柯帆,张峻,肖辰鹏,邢来君,李明春

微生物学通报 , 2013,
Abstract: Objective] The gene M-treS from Meiothermus ruber CBS-01 encodes a trehalose synthase of 962 amino acids, named M-TreS. To improve its catalytic activity, we constructed a method of molecular directed evolution, the sectionalized DNA shuffling. Methods] Through two PCR steps with two pairs of partially complementary primers, the M-treS gene was parted into two sections. After the two sections shuffled respectively, a whole gene was obtained through the complementarity of the primers. This method was more feasible, with higher mutability than normal DNA shuffling. Results] Mutants were obtained after one round of the sectionalized DNA shuffling, in combination with error-prone PCR. The best mutant enzyme contained 6 amino acid substitutions, whose catalytic activity and efficiency were 1.6-fold and 2-fold of that of the wild type, respectively. In the 6 amino acid substitutions, 5 were caused by homologous recombination, and one by error-prone PCR. Conclusion] This study indicates that the sectionalized DNA shuffling is an effective tool for molecular directed evolution of macromolecular proteins.
Molecular Evolution of Trehalose-6-Phosphate Synthase (TPS) Gene Family in Populus, Arabidopsis and Rice  [PDF]
Hai-Ling Yang, Yan-Jing Liu, Cai-Ling Wang, Qing-Yin Zeng
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0042438
Abstract: Trehalose-6-phosphate synthase (TPS) plays important roles in trehalose metabolism and signaling. Plant TPS proteins contain both a TPS and a trehalose-6-phosphate phosphatase (TPP) domain, which are coded by a multi-gene family. The plant TPS gene family has been divided into class I and class II. A previous study showed that the Populus, Arabidopsis, and rice genomes have seven class I and 27 class II TPS genes. In this study, we found that all class I TPS genes had 16 introns within the protein-coding region, whereas class II TPS genes had two introns. A significant sequence difference between the two classes of TPS proteins was observed by pairwise sequence comparisons of the 34 TPS proteins. A phylogenetic analysis revealed that at least seven TPS genes were present in the monocot–dicot common ancestor. Segmental duplications contributed significantly to the expansion of this gene family. At least five and three TPS genes were created by segmental duplication events in the Populus and rice genomes, respectively. Both the TPS and TPP domains of 34 TPS genes have evolved under purifying selection, but the selective constraint on the TPP domain was more relaxed than that on the TPS domain. Among 34 TPS genes from Populus, Arabidopsis, and rice, four class I TPS genes (AtTPS1, OsTPS1, PtTPS1, and PtTPS2) were under stronger purifying selection, whereas three Arabidopsis class I TPS genes (AtTPS2, 3, and 4) apparently evolved under relaxed selective constraint. Additionally, a reverse transcription polymerase chain reaction analysis showed the expression divergence of the TPS gene family in Populus, Arabidopsis, and rice under normal growth conditions and in response to stressors. Our findings provide new insights into the mechanisms of gene family expansion and functional evolution.
Analysis of TPS’s Actualization Problem in China and the Countermeasure  [cached]
Yuping Guo,Yachao Wang
Journal of Sustainable Development , 2009, DOI: 10.5539/jsd.v1n3p114
Abstract: As an effective production system, TPS (Toyota Production System) has been gone in for by numerous domestic and foreign factories recently. However, it is not effectively as in Japan actually, and many problems appear. This article analyses TPS’s domestic operational status, and put forward a set of reasonable countermeasures and proposals. This analysis will promote the TPS applicability research and guide the domestic enterprises to push TPS entirely.
Metagenomic islands of hyperhalophiles: the case of Salinibacter ruber
Lejla Pa?i?, Beltran Rodriguez-Mueller, Ana-Belen Martin-Cuadrado, Alex Mira, Forest Rohwer, Francisco Rodriguez-Valera
BMC Genomics , 2009, DOI: 10.1186/1471-2164-10-570
Abstract: Three regions of the sequenced isolate were scarcely represented in the metagenome thus appearing to vary among co-occurring S. ruber cells. These metagenomic islands showed evidence of extensive genomic corruption with atypically low GC content, low coding density, high numbers of pseudogenes and short hypothetical proteins. A detailed analysis of island gene content showed that the genes in metagenomic island 1 code for cell surface polysaccharides. The strain-specific genes of metagenomic island 2 were found to be involved in biosynthesis of cell wall polysaccharide components. Finally, metagenomic island 3 was rich in DNA related enzymes.The genomic organisation of S. ruber variable genomic regions showed a number of convergences with genomic islands of marine microbes studied, being largely involved in variable cell surface traits. This variation at the level of cell envelopes in an environment devoid of grazing pressure probably reflects a global strategy of bacteria to escape phage predation.Prokaryotic genomes are extraordinarily plastic entities and vary widely within the limits of a well defined species. In order to describe these large genetic reservoirs the pan-genome concept was introduced [1]. According to this concept, the species genome is composed of a core genome, containing genes present in all (or most) strains and a variable genome, containing genes present only in some strains.In some cases, this variation is concentrated in hypervariable sets of genes, known as genomic islands [2-4]. Genomic island genes are often involved in specific lifestyles [5,6], e.g. symbiosis or pathogenesis [7,8] and frequently have the hallmarks of horizontally transferred genetic material such as different GC content or codon usage [9,10]. However, very little is known about the dynamic processes that originate and maintain the large genomic variability found in closely related prokaryotic genomes.Metagenomics provides a new way to look at the dynamics and flexibili
Isolation and Biochemical Characterization of Rubelase, a Non-Hemorrhagic Elastase from Crotalus ruber ruber (Red Rattlesnake) Venom  [PDF]
Yumiko Komori,Kaname Sakai,Katsuyoshi Masuda,Toshiaki Nikai
Toxins , 2011, DOI: 10.3390/toxins3070900
Abstract: A novel non-hemorrhagic basic metalloprotease, rubelase, was isolated from the venom of Crotalus ruber ruber. Rubelase hydrolyzes succinyl-L-alanyl-L-alanyl-L-alanyl p-nitroanilide (STANA), a specific substrate for elastase, and the hydrolytic activity was inhibited by chelating agents. It also hydrolyzes collagen and fibrinogen. However, hemorrhagic activity was not observed. By ESI/Q-TOF and MALDI/TOF mass spectrometry combined with Edman sequencing procedure, the molecular mass of rubelase was determined to be 23,266 Da. Although its primary structure was similar to rubelysin (HT-2), a hemorrhagic metalloprotease isolated from the same snake venom, the circumstances surrounding putative zinc binding domain HEXXHXXGXXH were found to be different when the three-dimensional computer models of both metalloproteases were compared. The cytotoxic effects of rubelase and rubelysin on cultured endothelial and smooth muscle cells were also different, indicating that the substitution of several amino acid residues causes the changes of active-site conformation and cell preference.
新型沥青添加剂TPS的性能  [PDF]
张锐,黄晓明,侯曙光
交通运输工程学报 , 2006,
Abstract: 为了了解新型添加剂TPS的路用性能,进行了不同TPS掺量的沥青胶结料的针入度试验、软化点试验、稠度试验、延度及测力延度试验、弹性恢复试验及弯曲蠕变试验、直接拉伸试验,对加入TPS添加剂后的沥青混合料,进行了车辙试验、弯曲破坏试验、疲劳试验和冻融劈裂试验,并与SBS改性沥青混合料的部分试验结果进行了对比。结果表明添加TPS后,沥青胶结料的感温性、高温性与低温性及沥青混合料的高温稳定性、低温抗裂性、抗疲劳性和水稳定性都得到了较大程度的提高,添加TPS沥青混凝土具有良好的路用性能
Distribution, abundance and diversity of the extremely halophilic bacterium Salinibacter ruber
Josefa Antón, Arantxa Pe?a, Fernando Santos, Manuel Martínez-García, Philippe Schmitt-Kopplin, Ramon Rosselló-Mora
Aquatic Biosystems , 2008, DOI: 10.1186/1746-1448-4-15
Abstract: During the summer of 1998, in the course of a study focused on the identification by fluorescence in situ hybridization (FISH) of the then uncultured square archaeon, high proportions of Bacteria were detected by FISH in crystallizer ponds from solar salterns [1]. Although bacterial 16S rRNA gene sequences had been previously detected in these environments [2,3], that was the first report on high abundance of potentially active Bacteria, for which the candidatus name of "Salinibacter" was proposed. Shortly after, some of these Bacteria could be grown in pure culture and were characterized taxonomically [4,5]. The candidatus species was finally classified as a new genus and species, and named as Salinibacter ruber gen. nov. sp. nov. In these last few years a considerable advance in the knowledge of these microorganisms has been achieved [6-8] and even its genome has been completely sequenced and annotated [9].According to phylogenetic reconstructions based on the 16S rRNA gene [5] and on the inter-spacer region between the 16S and 23S rRNA genes [10] S. ruber can be affiliated with the phylum Bacteroidetes, being its closest related cultured organism Rhodothermus marinus, a thermophilic, slightly halophilic marine bacterium. The clade comprising R. marinus and S. ruber appeared as a deep branch within the phylum, placed close to the node of bifurcation of the superphylum that comprises Bacteroidetes and Chlorobi [11]. The phylogenetic position of S. ruber was further studied analyzing a total of 22 genes from the genome of S. ruber strain M31 [12]. All these genes had essential functions for the organism, were dispersed within the genome, and rendered a final alignment informative enough for phylogenetic reconstructions. Although single genes supported different topologies, the tree topology of concatenated genes was identical to that previously observed based on small subunit 16S rRNA gene analysis [12], a further confirmation of the validity of this gene for geneal
TPP争端解决机制及启示
 [PDF]

,
- , 2017,
Abstract: 摘要 作为美国实施“亚太再平衡”战略、重构全球经贸秩序、争夺规则主导权重要载体的TPP,其争端解决条款呈现出适用范围广泛、更加注重司法性、更加强调透明度和时效性,以及注重与其他国际体制的兼容性等特点,是WTO、NAFTA等现有国际经贸争端解决机制的“加强版”,也是“规则导向型”争端解决机制的“升级版”。但也要看到,TPP争端解决机制所涉主要内容及透射出价值早有端倪,在众多区域贸易协定中或隐或现,仅是绽放程度有所不同,区域贸易协定争端解决机制已呈趋同之势。我国在自贸协定谈判中要及时关注相关内容和主旨,借鉴以TPP为代表区域经贸协定争端解决制度设计,从法律文本和法律实践两个层面,完善我国自贸协定争端解决机制。
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