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Construction of a Metagenomic Library of Cellolytic Enrichment Culture and Cloning, Identification and Expression of a Novel Xylanase Gene

Author: MoXinChun
Tutor: FengJiaXun
School: Guangxi University
Course: Biochemistry and Molecular Biology
Keywords: Enrichment culture metagenomic library xylanase gene cloning identification expression
CLC: Q78
Type: Master's thesis
Year: 2007
Downloads: 17
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Abstract


A fiber and straw degrading enrichment culture was constructed according to theselective enrichment protocol. Metagenomic DNA was directly extracted from thisenrichment culture, ligated with cosmid vector pWEB::TNC to construct ametagenomic library containing about 12000 clones. Plasmids of 16 clones chosenrandomly from the library were extracted, digested with BamHI and analyzed byagarose gel electrophoresis. The result showed that each of them harbored differentforeign DNA fragments. And the large insert DNA fragment was 37 kb and thesmallest was 14 kb. The average size of the insert DNA fragment was 25.5 kb and thelibrary capacity of foreign DNA was 3.06×10~8bp. The restriction analysis of 16plasmids from library revealed that the cloned DNA fragments were very random.Two positive clones expressing exo-glucanase activity and six positive clonesexpressing cellulase activity were isolated from the library based on activity screeningmethod. Two positive clones expressing exo-glucanase activity designated as 3621and 70981, one positive clone expressing strongest cellulase activity designated as11210, were chosen for further analyzed. Three relative genes named umxyn10A,umgs1A and umcel9E respectively were identified by subcloning and sequencing.The ORF of umcel9E was 2,841 bp, which encoded an endoglucanase with 945amino acid residues. The Umcel9E shares highest homology with a1,4-beta-cellobiosidase (Accession number: EAM46961.1) from Clostridiumthermocellum ATCC27405 at 99% identity and 100% similarity. The ORF of umgs1Awas 1,707 bp, which encoded an enzyme showing activity against 4-MUC with 569amino acid residues. The Umgs1A shares highest homology with agamma-glutamylcysteine synthetase (Accession number: BAA14251.1) fromSaccharomyces cerevisiae at 99% identity and 99% similarity. The ORF of umxyn10A was 1,188 bp, which encoded a xylanase with 396 amino acid residues. The blastXresult show that the Umxyn10A shares highest homology with anendo-1,4-beta-xylanase (Accession number: AAZ56824.1) from Thermobifida fuscaYX at 62% identity and 78% similarity.One xylanase gene, umxyn10A, was overexpressed in E. coli and the purifiedrecombinant enzymes were characterized in more detail. The optima pH andtemperature of the recombinant Umxyn10A toward xylan was 6.5 and 75℃. And itwas stabled at pH5.0～7.0 and temperature blow 65℃. The Km and Vmax towardxylan (oat spelt) were 3.217 mg/ml and 217 U/mg protein, respectively. When usingρ-NPC as substrate, the optima pH and temperature of the recombinant Umxyn10Awas 6.5 and 70℃. The activity of recombinant Umxyn10A did not inhibite by most ofmetal irons and chelator EDTA. Only surfacant SDS can inhibite the activity ofrecombinant Umxyn10A toward xylan from oat spelt. These indicated thatUmxyn10A was’t a metal chelated enzyme. Substrate specificity analysis revealedthat Umxyn10A only catalyzedβ-1,4-glucan bond of saccharide. To investigate theactivities of Umxyn10A toward various cello-oligosaccharides, the hydrolysisproducts obtained from the substrates were qualitatively analyzed by TLC. Cellobiosewas not hydrolysed by Umxyn10A, and cellotriose was hydrolysed to producecellobiose and glucose, cellotetraose was mostly hydrolysed to cellobiose, anddegradation of cellopentaose produced both cellobiose and glucose. These indicatedUmxyn10A was an exo-action mode of xylanase.

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