A new approach for discovering cold-active enzymes in a cell mixture of pure-cultured bacteria

Abstract

To overcome the intrinsic problems of conventional approaches, such as the unavailability of source microorganisms in metagenomic libraries and the production of inactive aggregates, a new method was tested for discovering new enzymes (e.g. coldactive chitinase). A metagenome-like library was constructed using genomes extracted from a cell mixture of pure-cultured chitinolytic bacteria, followed by activity-based screening for <em>Escherichia coli</em> clones that exhibit chitinase activity on selective medium. Within one positive chitinolytic clone, one chitinase gene (<em>chi22718_III)</em> was detected and assigned to the arctic marine bacterium, <em>Pseudoalteromonas issachenkonii </em>PAMC 22718, by colony-PCR with <em>chi22718_III</em>-specific primers. When expressed in <em>E. coli</em>, recombinant R-Chi22718_III lost 85 % of its enzyme activity when pre-incubated at 40 ℃ for 1 h, whereas its mesophilic counterpart R-ChiK only lost 10 % of its activity under the same conditions indicating that R-Chi22718_III is thermolabile, a characteristic of cold-active enzymes.