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 pure-cultured chitinolytic bacteria, followed by activity-based screening for Escherichia 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.
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Acknowledgments
This work was financially supported by a grant from the Ministry of Oceans and Fisheries, Korea to KOPRI (project PM12030).
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Kim, D., Park, H.J., Kim, IC. et al. A new approach for discovering cold-active enzymes in a cell mixture of pure-cultured bacteria. Biotechnol Lett 36, 567–573 (2014). https://doi.org/10.1007/s10529-013-1384-2
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DOI: https://doi.org/10.1007/s10529-013-1384-2