Expression cloning, purification and characterization of a beta-1,4-mannanase from Aspergillus aculeatus

Biochem Mol Biol Int. 1994 Aug;33(5):917-25.

Abstract

A cDNA library from the filamentous fungus Aspergillus aculeatus was constructed in the yeast expression vector pYES2.0 and used to isolate 57 full length cDNA's encoding beta-1,4-mannanase by expression in S. cerevisiae. The positive clones were identified on agar plates containing 0.2% azurine dyed cross-linked mannan by the formation of blue halos around the colonies. All clones represented transcripts of the same mannanase gene (man1). The gene was sub-cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for overexpression and purification of the enzyme. The recombinant enzyme had a molecular weight of 45 kDa, an isoelectric point of pH 4.5, a pH optimum of pH 5.0 and a temperature optimum of 60-70 degrees.

MeSH terms

  • Amino Acid Sequence
  • Aspergillus / enzymology
  • Aspergillus / genetics*
  • Base Sequence
  • Cloning, Molecular
  • Gene Expression Regulation, Fungal
  • Gene Library
  • Genes, Fungal / genetics*
  • Isoelectric Point
  • Kinetics
  • Mannosidases / chemistry
  • Mannosidases / genetics*
  • Mannosidases / isolation & purification
  • Mannosidases / metabolism*
  • Molecular Sequence Data
  • Molecular Weight
  • Recombinant Fusion Proteins / biosynthesis
  • Sequence Alignment
  • Sequence Analysis, DNA

Substances

  • Recombinant Fusion Proteins
  • Mannosidases
  • endo-1,4-beta-D-mannanase

Associated data

  • GENBANK/L35487