Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses

<p>Abstract</p> <p>Background</p> <p><it>Neocallimastix patriciarum</it> is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (<it>GHs</it>) produced by this anaerobic fungus.</p> <p>Results</p> <p>We have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches to <it>N. patriciarum </it>to accelerate gene identification, enzyme classification and application in rice straw degradation. By conducting complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative <it>GH </it>contigs and classified them into 25 <it>GH</it> families. The secretome analysis identified four major enzymes involved in rice straw degradation: β-glucosidase, endo-1,4-β-xylanase, xylanase B and Cel48A exoglucanase. From the sequences of assembled contigs, we cloned 19 putative cellulase genes, including the <it>GH1</it>, <it>GH3</it>, <it>GH5</it>, <it>GH6</it>, <it>GH9</it>, <it>GH18</it>, <it>GH43 </it>and <it>GH48 </it>gene families, which were highly expressed in <it>N. patriciarum </it>cultures grown on different feedstocks.</p> <p>Conclusions</p> <p>These <it>GH </it>genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One β-glucosidase (W5-16143) and one exocellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.</p

Preliminary X-ray analysis of XC5848, a hypothetical ORFan protein with an Sm-like motif from Xanthomonas campestris

[[abstract]]XC5848, a hypothetical protein from the pathogenic bacterium Xanthomonas campestris that causes black rot, has been chosen as a potential target for the discovery of novel folds. It is unique to the Xanthomonas genus and has significant sequence identity mainly to corresponding proteins from the Xanthomonas genus. In this paper, the cloning, overexpression, purification and crystallization of the XC5848 protein are reported. The XC5848 crystals diffracted to a resolution of at least 1.68 angstrom. They belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 48.13, b = 51.62, c = 82.32 angstrom. Two molecules were found in each asymmetric unit. Preliminary structural studies nevertheless indicate that XC5848 belongs to the highly conserved Sm-like alpha-beta-beta-beta-beta fold. However, significant differences in sequence and structure were observed. It therefore represents a novel variant of the crucial Sm-like motif that is heavily involved in mRNA splicing and degradation.[[fileno]]2050134010044[[department]]生科

Expression and structure analysis of unknown function proteins XC5270、XC5848 and XC6398 in XCC by X-ray

近來眾多基因體的 DNA 序列陸續完成，使得結構蛋白質體學的研究日趨重要。蛋白質的三級結構決定其功能，因此決定一個未知功能蛋白質的結構可以幫助我們預測其功能。而 XCC 結構基因體計畫的最終目的為提供一個含有結構和功能註解的Xanthomonas campestris 結構基因庫，希望能找出一些新型的褶疊形式。目前估計所有蛋白質褶疊形式總數大約為一千種，但是目前只有約五百種不同的褶疊形式被發現。本論文主要研究的三個標的蛋白XC5270, XC5848 及XC6398 皆被歸類為未知功能蛋白。XC5270 利用生物資訊學做預測顯示其功能為一general stress protein，但其確切功能並不是很清楚，只知當細胞受到刺激或於不利的環境中，例如 heat shock、salt stress、oxidative stress 等等，它就會被誘導出以降低細胞所受到的傷害。XC5270 目前已經被大量表達，且篩選出適當的結晶條件，並成功製備含Se-Met 的蛋白，但其繞射圖譜上有多餘的點，造成在auto-indexing 時 peak picking 的錯誤，以致於數據無法最佳化。而 XC5848 以及XC6398 在預測上皆為conserved hypothetical protein，它們不屬於任何一個蛋白質 家族。XC5848 在 pET20b 的表現載體中可得到大量表現的可溶蛋白，但其表現出的蛋白未設計水解Due to the fast genome sequencing in recent year, the issue of structural genomicshas been becoming an important endeavor, due to the fact that protein structure can help determine its function. The final purpose of XCC structure genomics is to provide a Xanthomonas campestris protein database that contain structure and function annotation. In addition, it is also hoped that novel folds can be found by studying the XCC structural genomics. The three target proteins XC5270, XC5848 and XC6398 in this thesis are classified as unknown function proteins. XC5270 was predicted as a general stress protein through a bioinformatics study. Its precise function is not very clear, but when the cell is subject to unfavorable environments, such as heat shock, salt stress, oxidative stress, etc, it will be induced to protect cell from injury. XC5270 have been over-expressed, and screened for proper crystal formation. However, extra reflection points possibly due to the crystal twining have been observed that have caused problem in its data analysis. Better crystallization condition is necessary to prevent this problem.On the other hand, both XC5848 and XC6398 are classified as conserved hypothetical proteins. XC5848 could be over-expressed to get soluble protein using the expression vector pET20b. However, since no protease cutting site was engineered, the extra amino acid sequence at the XC5848 C-terminal end may have made its crystallization difficult. We have therefore ligated the XC5848 to a modified vector pTBSG1 to solve this problem. So far good crystals of Se-Met substituted proteins have been obtained and good diffraction pattern to a resolution of 2.0中文摘要........................................................................................................................... I Abstract.......................................................................................................................... III 縮寫檢索表................................................................................................................... VII 實驗儀器設備..............................................................................................................VIII 第一章 前言.....................................................................................................................1 第二章 材料方法.............................................................................................................5 2-1、標的蛋白之選取............................................................................................. 5 2-2、引子之設計與合成......................................................................................... 5 2-3、染色體 DNA 的抽取.................................................................................... 6 2-4、聚