Cloning, characterization and overproduction of nuclease S1 gene (nucS) from Aspergillus oryzae

Appl Microbiol Biotechnol. 1995 Dec;44(3-4):425-31. doi: 10.1007/BF00169939.

Abstract

The nuclease S1 gene (nucS) from Aspergillus oryzae was isolated using a polymerase-chain-reaction-amplified DNA fragment as a probe, and a 2.6-kb SalI-EcoRI fragment containing the nucS gene was sequenced. It was deduced that the nucS gene had two short introns, 49 and 50 nucleotides in length. The nucS gene had an open-reading frame of 963 base pairs and coded for a protein of 287 amino acid residues, comprising the signal peptide of 20 amino acids and a mature protein of 267 amino acids. The deduced amino acid sequence agreed well with the published amino acid sequence except for one substitution. Southern hybridization analysis showed that the nucS gene existed as a single copy in the A. oryzae chromosome. When the structural gene of nucS was fused with the promoter of the glaA gene and introduced into A. oryzae, the yield of secreted nuclease S1 increased about 100-fold compared with the recipient strain.

MeSH terms

  • Amino Acid Sequence
  • Aspergillus oryzae / enzymology
  • Aspergillus oryzae / genetics*
  • Base Sequence
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Fungal Proteins / biosynthesis
  • Fungal Proteins / genetics*
  • Fungal Proteins / isolation & purification
  • Genes, Fungal*
  • Glucan 1,4-alpha-Glucosidase / genetics
  • Molecular Sequence Data
  • Promoter Regions, Genetic
  • Recombinant Fusion Proteins / biosynthesis
  • Single-Strand Specific DNA and RNA Endonucleases / biosynthesis
  • Single-Strand Specific DNA and RNA Endonucleases / genetics*
  • Single-Strand Specific DNA and RNA Endonucleases / isolation & purification

Substances

  • Fungal Proteins
  • Recombinant Fusion Proteins
  • Single-Strand Specific DNA and RNA Endonucleases
  • Glucan 1,4-alpha-Glucosidase

Associated data

  • GENBANK/D45902