title: 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 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.
authors.affiliation: Division of Life Sciences, Korea Polar Research Institute, Incheon, 406-840, Korea, envimic@kopri.re.kr.
authors.forename: Dockyu
authors.initials: D
authors.lastname: Kim
authors.forename: Ha Ju
authors.initials: HJ
authors.lastname: Park
authors.forename: Il-Chan
authors.initials: IC
authors.lastname: Kim
authors.forename: Joung Han
authors.initials: JH
authors.lastname: Yim
mesh_terms: D002688:Chitinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008403:Mass Screening / Q000379:methods*; D054892:Metagenome; D008969:Molecular Sequence Data; D031161:Pseudoalteromonas / Q000235:genetics; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D002688:Chitinases