Cloning and Characterisation of an -amylase gene from the thermophilic fungus Thermomyces lanuginosus

Preben Rasmussen and Birgit Michelsen

Author address: 

DANISCO Biotechnology, DK-1001 Copenhagen K, Denmark

Abstract: 

Fungal -amylases have been used for many years in food industry, especially in starch and bakery industry. Fungal amylases have traditionally been fermented from Aspergillus oryzae. However this amylase is not very heat stable and have little if any activity during starch gelatinasation. Several thermophilic fungi, among these T lanuginosus, produces -amylases (1) with significant higher heat stability, unfortunately in rather low amounts. In order to study the -amylase from a thermophilic fungus we have cloned and characterised the gene encoding the -amylase from T lanuginosus . A PCR fragment of the gene was isolated by designing primers specific to conserved regions of fungal -amylases. The cloned fragment was used to screen a genomic library of T lanuginosus and 5 independent clones were found. The clones contained an ORF encoding a 493 residues long protein with a putative signal sequence of 18 residues. The ORF was interrupted by eight introns. Two peptides of purified -amylase was sequenced which confirmed the isolated gene encodes the correct enzyme. This ORF was expressed in Aspergillus niger resulting in high expression of -amylase in the transformants. The heterologous protein was purified and characterised biochemically, which showed that it was substantially identical to the native protein. In contrast to the heterologous protein the native protein was N-terminally blocked. The N-terminal sequence of the mature protein confirmed the putative identified signal sequence cleavage site. The cloning and expression of this gene in A. niger have given a sufficient and reliable source of T lanuginosus -mylase for characterisation and application tests. (1) Jensen, B. and Olsen J. (1992) Enzyme Microb. Technol. 14: 112-116
1996

abstract No: 

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Full conference title: 

3rd EUROPEAN CONFERENCE ON FUNGAL GENETICS
    • ECFG 3rd (1996)