title: Discovery of novel enzymes with industrial potential from a cold and alkaline environment by a combination of functional metagenomics and culturing.
abstract: BACKGROUND
  The use of cold-active enzymes has many advantages, including reduced energy consumption and easy inactivation. The ikaite columns of SW Greenland are permanently cold (4-6°C) and alkaline (above pH 10), and the microorganisms living there and their enzymes are adapted to these conditions. Since only a small fraction of the total microbial diversity can be cultured in the laboratory, a combined approach involving functional screening of a strain collection and a metagenomic library was undertaken for discovery of novel enzymes from the ikaite columns.
  
  
  RESULTS
  A strain collection with 322 cultured isolates was screened for enzymatic activities identifying a large number of enzyme producers, with a high re-discovery rate to previously characterized strains. A functional expression library established in Escherichia coli identified a number of novel cold-active enzymes. Both α-amylases and β-galactosidases were characterized in more detail with respect to temperature and pH profiles and one of the β-galactosidases, BGalI17E2, was able to hydrolyze lactose at 5°C. A metagenome sequence of the expression library indicated that the majority of enzymatic activities were not detected by functional expression. Phylogenetic analysis showed that different bacterial communities were targeted with the culture dependent and independent approaches and revealed the bias of multiple displacement amplification (MDA) of DNA isolated from complex microbial communities.
  
  
  CONCLUSIONS
  Many cold- and/or alkaline-active enzymes of industrial relevance were identified in the culture based approach and the majority of the enzyme-producing isolates were closely related to previously characterized strains. The function-based metagenomic approach, on the other hand, identified several enzymes (β-galactosidases, α-amylases and a phosphatase) with low homology to known sequences that were easily expressed in the production host E. coli. The β-galactosidase BGalI17E2 was able to hydrolyze lactose at low temperature, suggesting a possibly use in the dairy industry for this enzyme. The two different approaches complemented each other by targeting different microbial communities, highlighting the usefulness of combining methods for bioprospecting. Finally, we document here that ikaite columns constitute an important source of cold- and/or alkaline-active enzymes with industrial application potential.
authors.forename: Jan Kjølhede
authors.initials: JK
authors.lastname: Vester
authors.forename: Mikkel Andreas
authors.initials: MA
authors.lastname: Glaring
authors.affiliation: Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark. psg@plen.ku.dk.
authors.forename: Peter
authors.initials: P
authors.lastname: Stougaard
mesh_terms: D001419:Bacteria / Q000201:enzymology* / Q000235:genetics*; D003080:Cold Temperature; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D056186:Metagenomics; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000145:classification / Q000235:genetics; D017422:Sequence Analysis, DNA; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D012336:RNA, Ribosomal, 16S; D000516:alpha-Amylases; D001616:beta-Galactosidase