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

Cited 5 time in scopus
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Title
A new approach for discovering cold-active enzymes in a cell mixture of pure-cultured bacteria
Other Titles
순수배양한 세균의 세포혼합액에서 저온활성 효소를 발굴하기 위한 새로운 탐색 방법
Authors
Kim, Dockyu
Yim, Joung Han
Kim, Il-Chan
Park, Ha Ju
Keywords
Activity-based enzyme screen; Chitinase; Functional expression; Metagenomic library; Pseudoalteromonas issachenkonii
Issue Date
2014
Publisher
Springer
Citation
Kim, Dockyu, et al. 2014. "A new approach for discovering cold-active enzymes in a cell mixture of pure-cultured bacteria". BIOTECHNOLOGY LETTERS, 36(3): 567-573.
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.
URI
http://repository.kopri.re.kr/handle/201206/7248
DOI
http://dx.doi.org/10.1007/s10529-013-1384-2
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