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., cold-active chitinase). A metagenome-like library was constructed using genomes extracted from a cell mixture of purely cultured chitinolytic bacteria, followed by activity-based screening for E. coli clones that exhibit chitinase activity on selective medium. Within one positive chitinolytic clone, one chitinase gene (chi22718_III) was detected and assigned to the Arctic marine bacterium Pseudoalteromonas issachenkonii PAMC 22718 by colony-PCR with chi22718_III-specific primers. When expressed in E. coli, recombinant R-Chi22718_III lost 85% of its enzyme activity when pre-incubated at 40°C 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.