Expression cloning of fungal enzyme genes; a novel approach for efficient isolation of enzyme genes of industrial relevance.
Expression cloning is a relatively new method for fast and efficient cloning of enzyme genes from fungi that are known to make complex enzyme mixtures. In contrast to traditional cloning methods that are usually dependent on knowledge of at least a partial amino acid sequence in order to synthesize appropriate DNA probes or primers, the expression cloning method solely relies on access to reliable and sensitive enzyme assays. A representative expression cDNA library is made in Saccharomyces cerevisiae form the donor strain and relevant cDNA clones are detected directly based on the encoded enzyme activity. Thus, time-consuming enzyme purification and characterization steps are avoided. The method has been applied on the characterization of extracellular enzyme genes from the filamentous fungus Aspergillus aculeatus and has resulted in the isolation of 20 different enzyme genes such as endo-glucanases, xylanases, pectinases, proteases, hemicellulases and rhamnogalacturonan-degrading enzymes. All enzymes have been expressed in Aspergillus oryzae, purified and characterized. In the present review a description of the expression cloning technique will be given as well as examples of how the technique has been used in the exploration and characterization of a commercial enzyme product that is known to consist of a complex mixture of more than 25 different enzyme activities.
Dalbøge
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Enzyme Business. Novo Nordisk AIS, Bagsvard, Denmark. hda@novo.dk
D001230:Aspergillus / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D005658:Fungi / Q000201:enzymology*; D005800:Genes, Fungal; D015003:Yeasts / Q000235:genetics
D018076:DNA, Complementary