Expression of the Starch-binding Domain of Glucoamylase from Aspergillus niger and Determination of its Structure

Marie-Francoise Le Gal-Coeffet1,2, David Archer1, Kay Sorimach1, Amanda Jacks2 and Mike Williamson2

Author address: 

1Institute of Food Research, Norwich Research Park, Norwich, UK. 2Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK

Abstract: 

The starch-binding domain (SBD) of glucoamylase 1 (GI) of Aspergillus niger is a separate domain required for the binding of glucoamylase to granular starch. SBD has been produced by proteolysis of GI but the yields were poor and the method was time and labour consuming. An expression vector, using the glucoamylase sequence (residues 1-498) as carrier in a fusion construct has been used to produce from Aspergillus niger the starchbinding domain on its own. The endoproteolytic cleavage recognition site, Lys-Arg, introduced between G498 and the SBD sequences, was correctly processed in vivo. Yields of secreted SBD in different culture media have been studied and up to 300mg/l achieved in shake flasks. Biochemical and physical analysis showed that the engineered SBD is functionally active and 2 mol cyclodextrin/mol protein are bound with an affinity very similar to the proteolytically-derived SBD. The threonine residues 510, 511 and 513 are glycosylated with an average of three mannose residue per SBD molecule. 15N labelled SBD has been successfully produced at 20-40 mg/l with a specific activity of >99% for high resolution NMR analysis. The labelled protein was used to determine NMR ambiguities. The secondary structure has been resolved and it consists of one parallel and five antiparallel pairs of B-strands forming two B-sheets. The three dimensional structure of the SBD has been also completed. In order to study the molecular basis of starch binding, mutagenesis of specific amino acid residues of the SBD, which are thought to be involved in the binding of polysaccharides, has been undertaken. Binding affinity studies with the mutated SBDs are underway
1996

abstract No: 

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Full conference title: 

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