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

Biotechnol Lett. 2014 Mar;36(3):567-73. doi: 10.1007/s10529-013-1384-2. Epub 2013 Oct 25.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chitinases / chemistry
  • Chitinases / genetics
  • Chitinases / metabolism*
  • Cloning, Molecular
  • Enzyme Stability
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Mass Screening / methods*
  • Metagenome*
  • Molecular Sequence Data
  • Pseudoalteromonas / genetics
  • Sequence Analysis, DNA
  • Temperature

Substances

  • Chitinases

Associated data

  • GENBANK/KF574005