1,720,957 research outputs found
Cloning and Expression of Manganese Peroxidase Genes from Ganoderma spp.
No full text靈芝屬(Ganoderma spp.)是一種白腐型真菌，具有各種可分解木材的外泌酵素，包括纖維素水解酵素(cellulase)和半纖維素水解酵素(hemicellulase),、果膠質水解酵素(pectinase)、漆氧化酶(laccase)、木質素過氧化酶(lignin peroxidase)和錳過氧化酶(manganese peroxidase)。其中錳過氧化酶是一種含有血基質(heme)的酵素，在二價錳離子以及過氧化氫存在的環境中能將酚類多環物質氧化分解，白腐型真菌利用此酵素分解木質素結構使其菌絲能有效深入木質纖維中分解纖維素等多醣以獲得養分。本實驗以含有ABTS的培養皿測試十株靈芝屬菌株的胞外酵素，發現所測試靈芝屬真菌都有可分解ABTS(2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid))的外泌酵素。並利用Genome Walking、五端RACE和PCR增幅技術自台灣靈芝(G. formosanum 0109, BZ)、新日本靈芝(G. neo-japonicum 0813, VZ)和南方靈芝(G. australe 0705)中選殖出六段新的錳過氧化酶基因序列。以RT-PCR增幅基因首尾端具有限制酶切位的台灣靈芝0109錳過氧化酶的cDNA，並將此基因轉殖入大腸桿菌(Escherichia coli)以及嗜甲醇畢赤氏酵母(Pichia methanolica)進行異源表達，得到在C端接有六個組胺酸(histidine)的錳過氧化酶以利後續純化的重組蛋白。Lignin, component of plant secondary cell wall, binds to hemicellulose by covalent bonds and defends bacteria or fungi invading into the core of the wood. Lignin is a complicated polymer composed of aromatic compounds, which are ferulic acid, sinapic acid and p-coumaric acid etc. In paper pulping industry, lignin is hard-degradable and influences the brightness of paper. Generally, people use concentrated acid or alkali to precipitate or dissolve lignin. In recent study, the paper would be brighter and whiter by using manganese peroxidase in pulping procedure.
Manganese peroxidase, which was first isolated from white rot fungi Phanerochaete chrysosporium by Glenn and Gold in 1985, belongs to heme-containing peroxidase family. This extracellular enzyme is a glycoprotein with five disulfide bonds in general and contains two calcium ions in the structure. When it catalyses the substrates, hydrogen peroxide which produced by glyoxal oxidase or aryl-alcohol oxidase is necessary as electron donor. Mn(II) would be oxidized to Mn(III) by heme-oxygen radical complex. Mn(III) cations would be stabilized by alfa-hydroxyl acids, such as succinic acid or lactic acid, and oxidize lignin or lignin-like compounds.
Manganese peroxidase has been found in many of white rot fungi, including Trametes versicolor, Pleurotus ostreatus, Armillaria mellea, etc, however, the studies of manganese peroxidase from Ganoderma spp. are few and most of them were focus on preotein level. The only published cDNA sequence was from G. applanatum. In this study, we cloned six new sequences of manganese peroxidase from G. formosanum, G. neo-japonicum and G. australe. Furthermore, we expressed the manganese peroxidase of G. formosanum in Escherichia coli and Pichia methanolica and produced recombinant enzyme.目錄
中文摘要……………………………………………..………………….V
英文摘要…………………………………………..…………...……….VI
表目錄…………………………………………..………………….......VII
圖目錄………………………………………..………...…………......VIII
第一章 前言…………………………………………………..…..……1
1.木質素………………………………………………………..…..…….1
1.1.木質素的來源及構造………………………………………..………1
1.2木質素在構型上的相似物…………………………………..………5
1.3木質素及其相似物對工業環境或生命之負面影響………..………5
1.4分解木質素的方法…………………………………………..…..…...7
2.錳過氧化酶…………………………………………………..…..…….7
2.1錳過氧化酶的研究歷史……………………………………..…..…...7
2.2與錳過氧化酶功能相似的酵素……………………………..…..…...9
2.3錳過氧化酶的特性及適用條件……………………………..……….9
2.4錳過氧化酶的基質和應用…………………………………..…..….14
2.5已開發之錳過氧化酶重組表現系統………………………..…..….18
2.5.1同源表達系統..………………………………………………..…..18
2.5.2異源表達系統..………………………………………………..…..19
2.5.2.1原核表達系統..………….…………………………………..…..19
2.5.2.2真核表達系統.…………….……………………..…………..….20
3.靈芝屬真菌..…………………………………………….………..…..22
3.1靈芝屬真菌的特性…………………………………….………..…..22
3.2靈芝屬真菌對植物的病害………………..…………….………..…24
3.3靈芝屬真菌木質分解酵素的研究………..…………….………..…24
4.異源表達系統……………………………..……………….……..…..26
4.1原核表達系統…………………………………………….……..…..26
4.2真核表達系統…………………………………………….……..…..26
4.2.1嗜甲醇畢赤氏酵母表達系統…………………….…….……..…..26
5.研究動機與目的…………………………………………….……..…27
第二章 材料與方法……………………………………….……..…...28
1.實驗材料…………………………………………………….……..…28
1.1實驗菌株………………………………………………….……..…..28
1.2實驗質體………………………………………………….……..…..30
1.3菌種保存………………………………………………….……..…..30
2.實驗流程及方法…………………………………………...……..…..30
2.1靈芝屬真菌ABTS分解能力測定..........………………...……..…..30
2.1.1培養皿測試…………………………………………......……..…..30
2.1.2胞外液酵素活性測試………………………………......……..….32
2.2錳過氧化酶基因選殖.........................................................................33
2.2.1真菌DNA製備……………………………………….......…..…..33
2.2.2錳過氧化酶片段聚合酶連鎖反應增幅………………......…..…..34
2.2.3 PCR產物純化及定序…………………………………….......…..34
2.2.4真菌RNA製備……….………………………………….......……34
2.2.5各菌株錳過氧化酶選殖方法…..…………….......………..…..….35
2.2.5.1台灣靈芝(G. formosanum 0109, BZ)錳過氧化酶基因選殖……35
2.2.5.1.1 Rapid Genome Walking……………………………………….35
2.2.5.1.2片段cDNA合成………………………………………...……36
2.2.5.1.3五端RACE ( Rapid Amplification of cDNA Ends)……..……36
2.2.5.1.4 台灣靈芝(G. formosanum BZ)錳過氧化酶基因選殖……….37
2.2.5.2 南方靈芝(G. australe 0705)錳過氧化酶基因選殖……………37
2.2.5.3新日本靈芝(G. neo-japonicum 0813, VZ)錳過氧化酶基因選殖………………………………………………………………………..37
2.2.6胺基酸序列分析…………………………………………………..38
2.3錳過氧化酶基因之異源表達系統建構及表達......……..………….42
2.3.1.1大腸桿菌表達系統建構………………….......….…..………….42
2.3.1.2以大腸桿菌表達錳過氧化酶…………….......…...…………….42
2.3.2.1嗜甲醇畢赤氏酵母表達系統建構……….....….…..…..……….43
2.3.2.2以嗜甲醇畢赤氏酵母表達錳過氧化酶….......…...…..…..…….43
2.3.2.2.1培養皿測試………………………..…….......……..………….44
2.3.3蛋白質分析……………………………….......……..……...…….44
2.3.3.1活性測試……………………………..........……..………..…….44
2.3.3.2 蛋白質定量…………….………………………………………44
2.3.3.3十二烷基磺酸鈉聚丙烯醯胺膠體電泳...…...…….……...…….44
2.3.3.4.西方雜合分析………………………….….......……..…...…….45
2.3.3.5重組蛋白之純化……………………………...…………………45
第三章 結果…………………………………….......……..…....…….46
1.靈芝屬真菌分解ABTS能力之初步測定………….......……....…….46
1.1培養皿測試………………………….......……..…...……………….46
1.2 菌絲體胞外酵素活性測定………….......……..…......……………50
2. 靈芝屬真菌錳過氧化酶基因選殖…….......……..…...…….………50
2.1 錳過氧化酶基因片段選殖………….......……..…...……...………51
2.2 錳過氧化酶轉譯區間選殖………….......……..…...……...………51
2.2.1 台灣靈芝0109基因選殖…………………...……………………51
2.2.1.1Genome Walking…………..…………….………………………51
2.2.1.2 RT-PCR和五端RACE…..…………….………………………..54
2.2.1.3台灣靈芝BZ錳過氧化酶基因選殖………….…………………54
2.3.2 南方靈芝0705錳過氧化酶基因選殖…………….…..…………57
2.3.3 新日本靈芝0813, VZ錳過氧化酶基因選殖….…...……………57
2.3.4 錳過氧化酶片段基因分析………….………………..…….……60
2.3.5 南方靈芝0705和台灣靈芝0109錳過氧化酶轉譯區間比較..…60
2.3.6 靈芝屬物種錳過氧化酶胺基酸序列和其他物種之比較…....…69
3.靈芝錳過氧化酶的異源表達………….………………..……..……..72
3.1大腸桿菌表達系統………….………………..……..…………..…..72
3.1.1表達載體建構………….………………..……..…………..……...72
3.1.2 目標蛋白表達………….………………..……..…………..…….72
3.2嗜甲醇畢赤氏酵母菌表達系統……………..……..……………….77
3.2.1表達載體建構………….………………..……..…………..……...77
3.2.2目標蛋白表達………….………………..……..…………..……...77
第四章 綜合討論與結果…………..……..……….…………..……...80
第五章 未來展望……………………………………………………..84
參考文獻………….……………………..……..………………..……...8
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Microbial diversity, activity, and ecology of a hypersaline high Arctic spring system
No full textThe Lost Hammer (LH) spring, located on Axel Heiberg Island in the Canadian High Arctic, is the coldest and saltiest terrestrial spring discovered to date. It is characterized by perennial discharges of subzero temperatures (-5°C), hypersalinity (24% salinity), along with reducing (≈-165 mV), microoxic, and oligotrophic conditions. It is rich in sulfates (10.0% w/w), dissolved H2S/sulfides (up to 25 ppm), ammonia (≈381 μM), and methane (11.1 g d-1). The LH spring system contains the outlet and the outflow channel. In the initial study of the LH channel sediment, the results determined the microbial abundance by using fluorescent microscopy in the channel sediment; also, the study characterized the cultured representatives and confirmed that most of these isolates are halotolerant and psychrotolerant microorganisms. The mineralization assays on the LH channel sediment revealed that the heterotrophic microorganisms remained activity down to -20°C. To determine the total microbial communities inhabiting the LH spring system, the study demonstrated the microbial 16S rRNA genes and the active 16S rDNA profiles for different sampling locations, including the outlet, channel and the adjacent tundra. We identified that the Bacteria from the five phyla (Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes, and Cyanobacteria) were the dominant bacterial groups at the LH spring system. In the archaeal communities, microorganisms affiliated with three phyla (Euryarchaeota, Crenarchaeota, and Thaumarchaeota) were identified. To determine its total functional and genetic potential, we performed metagenomic analysis of the LH spring outlet microbial community. Reconstruction of the enzyme pathways responsible for bacterial nitrification/denitrification/ammonification and sulfate reduction appeared nearly complete in the metagenomic dataset. Stress-response genes for adapting to cold, osmotic stress, and oxidative stress were also abundant in the metagenome. Comparing functional community composition of the LH spring to metagenomes from other saline/subzero environments revealed a close association between LH and another Canadian High Arctic permafrost environment, particularly in genes related to sulfur metabolism and dormancy. To identify the abundance and the presence of the featured genes (amoA and hcd) of Thaumarchaea at the LH spring system, we performed qPCR to assess their abundance. A phylogenetic analysis was performed using the putative amino acid sequences of these genes to identify their phylogenetic affiliation. The copy numbers of Thaumarchaeal amoA and hcd genes in LH channel sediment and the adjacent tundra were roughly 10 to a hundred-folds less than those reported in other environments. The phylogenetic tree of amoA showed similar patterns of grouping as the analysis done by 16S rRNA. This thesis demonstrates the microbial ecology, diversity and activity at the LH spring system and provides knowledge for the microbiology studies on cryo- and hypersaline environments.La source de Lost Hammer (LH), située sur l'ile d'Axel Heiberg dans le Grand Nord Canadien, est la source la plus froide et la plus salée découverte à ce jour sur terre. Elle est caractérisée par des décharges pérennes de températures sous zéro (-5°C), par ses conditions hyper salines (salinité de 24%), réductrices (≈-165 mV), microoxiques, et oligotrophiques. Elle est aussi riche en sulfates (10.0% w/w), en H2S/sulfites dissouts, en ammoniac (≈381 μM), et en methane (11.1 g d-1). Le système de la Source de LH contient la sortie et le canal de sortie. L'étude originale des sédiments du canal détermina l'abondance de microbes avec des techniques de microscopie fluorescente. L'étude caractérisa aussi les cultures représentatives et confirma que la majorité des isolats sont des microorganismes halotolérants et psychrotolérants. Les tests de minéralisation des sédiments du canal de LH ont révélés que les microorganismes hétérotrophes restent actifs jusqu'à -20°C. Pour determiner la communauté totale vivant dans le système de la source de LH, l'étude investigua les profils de l'ARNr 16S ainsi que l'ARNr 16S actif microbien pour différents endroits d'échantillonnage, incluant la sortie, le canal, et la toundra adjacente. Nous avons identifié que les bactéries de cinq phylums (Bacteroidetes, protéobactéries, Actinobactéries, Firmicutes et Cyanobactéries) étaient les groupes de bactéries dominantes dans ce système. Dans les communautés archées, des microorganismes affiliés avec trois phylums (Euryarchaeota, Crenarchaeota et Thaumarchaeota) ont été identifiés. Pour déterminer son potentiel fonctionel et génétique total, nous avons performé l'analyse métagénomique de la communauté microbienne de la sortie de la source de LH. La reconstruction des voies enzymatiques responsables pour la nitrification/dénitrification/ammonification bactériennes et pour la réduction du sulfate apparut presque complète dans la banque de donnés métagnénomique. Les gènes de réponse au stress pour l'adaptation au froid, pour les chocs osmotiques et pour le stress oxydatif étaient aussi abondants dans le métagénome. En comparant la composition des communautés fonctionnelles du métagénome de la Source de LH, avec d'autres environnements salins et sous zéro, a révélé une association entre LH et un autre environnement du permafrost du Grand Nord Canadien, particulièrement dans les gènes reliés au métabolisme de sulfure et de dormance. Pour identifier l'abondance et la présence de gènes d'intérêt (amoA et hcd) de Thaumarchaea dans le système, nous avons performé les expériences qPCR. Une analyse phylogénique a aussi été faite pour identifier leur affiliation phylogénique en utilisant les séquences probables d'acides aminés de ces gènes. Le nombre de copies des gènes amoA et hcd de Thaumarchaea dans les sédiments du canal et dans la toundra adjacente étaient environ dix à cent fois moins que ceux rapportés dans d'autres environnements. L'arbre phylogénique de amoA a démontré des motifs similaires de regroupement à ceux de l'analyse faite avec r16S rADN. Cette thèse démontre l'écologie microbienne, la diversité et l'activité du système de la Source de LH, et apporte un savoir pour les études de microbiologie sur des environnements froids et hypersalins
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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