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. 1993 May 15;292(Pt 1):197–202. doi: 10.1042/bj2920197

Production, purification and characterization of the catalytic domain of glucoamylase from Aspergillus niger.

B Stoffer 1, T P Frandsen 1, P K Busk 1, P Schneider 1, I Svendsen 1, B Svensson 1
PMCID: PMC1134288  PMID: 8503847

Abstract

The catalytic domain of glucoamylases G1 and G2 from Aspergillus niger is produced in vitro in high yield by limited proteolysis using either subtilisin Novo or subtilisin Carlsberg. Purification by affinity chromatography on an acarbose-Sepharose column followed by ion-exchange chromatography on HiLoad Q-Sepharose leads to separation of a number of structurally closely related forms of domain. The cleavage occurs primarily between Val-470 and Ala-471 as indicated by C-terminal sequencing, whereas the N-terminus is intact. Subtilisin Carlsberg, in addition, produces a type of domain which is hydrolysed before Ser-444, an O-glycosylated residue. This leaves the fragment Ser-444-Val-470 disulphide-bonded to the large N-terminal part of the catalytic domain. Subtilisin Novo, in contrast, tends to yield a minor fraction of forms extending approx. 30-40 amino-acid residues beyond Val-470. The thermostability is essentially the same for the single-chain catalytic domain and the original glucoamylases G1 and G2, whereas the catalytic domain cut between Ser-443 and Ser-444 is less thermostable. For both types of domain the kinetic parameters, Km and kcat., for hydrolysis of maltose are very close to the values found for glucoamylases G1 and G2.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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