Secretion, purification, and characterisation of barley alpha-amylase produced by heterologous gene expression in Aspergillus niger

Appl Microbiol Biotechnol. 1998 Apr;49(4):385-92. doi: 10.1007/s002530051187.


Efficient production of recombinant barley alpha-amylase has been achieved in Aspergillus niger. The cDNA encoding alpha-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for alpha-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44,960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley alpha-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol beta-D-maltoheptaoside and amylose (degree of polymerisation = 17). Barley alpha-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus niger / enzymology*
  • Aspergillus niger / genetics
  • Blotting, Western
  • Electrophoresis, Polyacrylamide Gel
  • Genetic Vectors
  • Hordeum / enzymology*
  • Polymerase Chain Reaction
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Sequence Analysis
  • Transformation, Genetic
  • alpha-Amylases / genetics
  • alpha-Amylases / isolation & purification
  • alpha-Amylases / metabolism*


  • Recombinant Fusion Proteins
  • alpha-Amylases