Construction Of A Metagenomic Library Of Uncultured Microorganisms From Compost And Cloning, Identification And Expression Of Novel Xylanase Genes

Cells of uncultured microorganisms from self-made compost were isolated by differential centrifugation, metagenomic DNA of which was extracted, end-blunted and ligated with cosmid vector of pWEB::TNC to construct a metagenomic library containing about 50, 000 clones. Plasmids of 14 clones chosen randomly from the library were extracted, digested with BamHI and analyzed by agarose gel electrophoresis. The result showed that each of them contained an insert DNA fragment. The largest insert fragment was 45 kb and the smallest was 25 kb. The average size of the insert DNA was 35 kb and the capacity of foreign DNA of the library was 1.8×10~6kb (1.8 Gb). The restriction patterns of 14 plasmids from library were different with each other, indicating that the cloned DNA fragments were very random.The library was screened for xylanase activity and two xylanase-expressing clones were isolated which were designated as pGXN1050 and pGXN1051. The xylanase genes on the two clones were subcloned, sequenced and analyzed. Results showed that the putative xylanase geneumxynllA on pGXN1050 had a 771 bp ORF encoding a product with 257 amino acids sharing 46% identity and 57% similarity with endo-1,4-beta-xylanase of Cellvibrio mixtus (GenBank accession No. Z48925.1). The putative xylanase gene umxynl IB on pGXN1051 had a 723 bp ORF, encoding a product with 241 amino acids sharing 73% identity and 80% similarity with endo-l,4-beta-xylanase of Cellvibrio mixtus (GenBank accession No. Z48925.1). Domain analysis with SMART tools showed that UmxynllA and Umxynl IB were members of family 11 glycosyl hydrolase. A comparison analysis of amino acids sequences of umxynllA and umxynllB with BLAST 2 SEQUENCES showed that they shared 45% identity and 59% similarity. Phylogenetic analysis showed that umxynllA and umxynllB might come from closely-related species of Cellvibrio. A DNA sequence containing the DNA region encoding the catalytic domain of Umxyn11B was amplified by PCR and cloned into E. coli expression vector pET30a (+), resulting in an expression plasmid pGXN11B. SDS-PAGE analysis revealed that pGXN11B could express in E. coli BL21 (DE3) pLysS and the expression product was about 25.98 kDa. The conditions for umxynllB expression in E.coli BL21 (DE3) pLysS had been investigated.