SECRETION, PURIFICATION, AND CHARACTERIZATION OF BARLEY ALPHA-AMYLASEPRODUCED BY HETEROLOGOUS GENE-EXPRESSION IN ASPERGILLUS-NIGER

Efficient production of recombinant barley alpha-amylase has been achi eved in Aspergillus niger. The cDNA encoding alpha-amylase isozyme 1 ( AMY1) and its signal peptide was placed under the control of the Asper gillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promote r and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for alpha-amylase activity and l ow protease activity. The recombinant AMY1 (reAMY1) was purified to ho mogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of th e recombinant protein indicated that the endogenous plant signal pepti de is correctly processed in A. niger. Electrospray ionisation/mass sp ectrometry gave a molecular mass for the dominant form of 44960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylati on apparently did not occur. The activities of recombinant and native barley alpha-amylases are very similar towards insoluble and soluble s tarch as well as 2-chloro-4-nitrophenol beta-D-maltoheptaoside and amy lose (degree of polymerisation = 17). Barley alpha-amylase is the firs t plant protein efficiently secreted and correctly processed by A. nig er using its own signal sequence.