Construction, characterization, and use of small-insert gene banks of DNA isolated from soil and enrichment cultures for the recovery of novel amidases.
To obtain new amidases of biocatalytic relevance, we used microorganisms indigenous to different types of soil and sediment as a source of DNA for the construction of environmental gene banks, following two different strategies. In one case, DNA was isolated from soil without preceding cultivation to preserve a high degree of (phylo)genetic diversity. Alternatively, DNA samples were obtained from enrichment cultures, which is thought to reduce the number of clones required to find a target enzyme. To selectively sustain the growth of organisms exhibiting amidase activity, cultures were supplied with a single amide or a mixture of different aromatic and non-aromatic acetamide and glycine amide derivatives as the only nitrogen source. Metagenomic DNA was cloned into a high-copy plasmid vector and transferred to E. coli, and the resulting gene banks were searched for positives by growth selection. In this way, we isolated a number of recombinant E. coli strains with a stable phenotype, each expressing an amidase with a distinct substrate profile. One of these clones was found to produce a new and highly active penicillin amidase, a promising biocatalyst that may allow higher yields in the enzymatic synthesis of beta-lactam antibiotics.
Gabor
Esther M
EM
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands.
de Vries
Erik J
EJ
Janssen
Dick B
DB
D000081:Acetamides / Q000737:chemistry; D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D040461:Gene Components; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D019015:Geologic Sediments / Q000382:microbiology*; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D009426:Netherlands; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D000081:Acetamides; D017931:DNA Primers; C018556:glycine amide; D000581:Amidohydrolases; D005998:Glycine