Abstract
Efficient production of recombinant barley α-amylase has been achieved in Aspergillus niger. The cDNA encoding α-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 α-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 α-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol β-d-maltoheptaoside and amylose (degree of polymerisation = 17). Barley α-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence.
Similar content being viewed by others
Author information
Authors and Affiliations
Additional information
Received: 22 August 1997 / Received revision: 21 November 1997 / Accepted: 29 November 1997
Rights and permissions
About this article
Cite this article
Juge, N., Svensson, B. & Williamson, G. Secretion, purification, and characterisation of barley α-amylase produced by heterologous gene expression in Aspergillus niger . Appl Microbiol Biotechnol 49, 385–392 (1998). https://doi.org/10.1007/s002530051187
Issue Date:
DOI: https://doi.org/10.1007/s002530051187