초록
Filamentous fungi have increasingly been used as hosts for heterologous protein production because of their high secretion capability and ability to add eukaryotic post-translational modifications. In this study, a novel uracil-deficient Aspergillus transformation system, which was based on an orotate phosphoribosyltransferase (pyrF) nutritional selection marker, was discovered. Additionally, a universal, purify-able vector that directed genes into the Aspergillus host strain was engineered. A genomic DNA segment encoding a novel α-amylase was isolated from the psychrotolerant fungus Geomyces pannorum and the open reading frame was determined, deduced 497 amino acids. G. pannorum α-amylase was then expressed in the newly constructed Aspergillus oryzae system, with an amylase activity reaching 958U/ml. It was purified to electrophoretic homogeneity and has a molecular mass of approximately 54kDa. The enzyme exhibited an optimal activity at pH 5.0 and 40<SUP>o</SUP>C and retained over 20% of maximal activity over the temperature range 0-20<SUP>o</SUP>C. To our knowledge, this report is the first of the heterologous expression of a cold-adapted enzyme in filamentous fungi. G. pannorum α-amylase is an economical amylase with many potential applications.