Identification of novel glycosyl hydrolases with cellulolytic activity against crystalline cellulose from metagenomic libraries constructed from bacterial enrichment cultures

To obtain cellulases that are capable of degrading crystalline cellulose and cedar wood, metagenomic libraries were constructed from raw soil sample which was covered to pile of cedar wood sawdust or from its enrichment cultures. The efficiency of screening of metagenomic library was improved more than 3 times by repeating enrichment cultivation using crystalline cellulose as a carbon source, compared with the library constructed from raw soil. Four cellulase genes were obtained from the metagenomic libraries that were constructed from the total genome extracted from an enrichment culture that used crystalline cellulose as a carbon source. A cellulase gene and a xylanase gene were obtained from the enrichment culture that used unbleached kraft pulp as a carbon source. The culture supernatants of Escherichia coli expressing three clones that were derived from the enrichment culture that used crystalline cellulose showed activity against crystalline cellulose. In addition, these three enzyme solutions generated a reducing sugar from cedar wood powder. From these results, the construction of a metagenomic library from cultures that were repetition enriched using crystalline cellulose demonstrated that this technique is a powerful tool for obtaining cellulases that have activity toward crystalline cellulose.