Discovery of (hemi-) cellulase genes in a metagenomic library from a biogas digester using 454 pyrosequencing
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APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Discovery of (hemi-) cellulase genes in a metagenomic library from a biogas digester using 454 pyrosequencing Xing Yan & Alei Geng & Jun Zhang & Yongjun Wei & Lei Zhang & Changli Qian & Qianfu Wang & Shengyue Wang & Zhihua Zhou
Received: 4 February 2013 / Revised: 10 April 2013 / Accepted: 13 April 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract In this study, 341, 246, and 386 positive clones with endo-β-1,4-glucanase, β-glucosidase, and endo-β-1,4xylanase activities, respectively, were identified by screening from a metagenomic fosmid library constructed from a biogas digester. Subsequently, pools of 4, 10, and 16 positive clones were subjected to 454 pyrosequencing in different subruns. In total, 21 unique glycosyl hydrolase (GH) genes were predicted by bioinformatic analysis, which showed similarities to their nearest neighbors from 39 % to 72 %. In addition to bioinformatics prediction, nine GH genes were expressed and purified to identify their activity with four kinds of substrates. The activities of the most expressed proteins were consistent with their annotation based on bioinformatics prediction; however, three GH genes belonging to the GH5 family showed different Xing Yan and Alei Geng contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00253-013-4927-5) contains supplementary material, which is available to authorized users. X. Yan : A. Geng : J. Zhang : Y. Wei : L. Zhang : C. Qian : Q. Wang : Z. Zhou (*) Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Fenglin Rd 300, Shanghai 200032, China e-mail: [email protected] A. Geng Biofuels institute, School of the Environment, Jiangsu University, Zhenjiang 212013 Jiangsu, China J. Zhang The College of Life Sciences, Northwest University, Xi’an 710069, China S. Wang Chinese National Human Genome Center at Shanghai, Shanghai 201203, China
activities from their annotation. An efficient acidic cellulase En1 had an optimal condition at 55 °C, pH 5.5, with a specific activity toward carboxymethylcellulose at 118 U/mg and Km at 12.8 g/L. This study demonstrated that there are diverse GHs in the biogas digester system with potential industrial application in lignocellulose hydrolysis, and their activities should be investigated with different substrates before their application. Additionally, pool sequencing of positive fosmid clones might be a costeffective approach to obtain functional genes from metagenomic libraries. Keywords 454 Pyrosequencing . Biogas digester . Fosmid library . Glycosyl hydrolase . Metagenome
Introduction The global demand for energy and chemicals is increasing, while reserves of fossil fuels are limited. Lignocellulosic biomass is expected to be a renewable resource capable of producing fuels and chemicals through biorefinery processes (Demain et al. 2005). The rate and cost of lignocellulose hydrolysis by enzymes
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