PRODUCTION, PURIFICATION AND CHARACTERIZATION OF THE CATALYTIC DOMAINOF GLUCOAMYLASE FROM ASPERGILLUS-NIGER

The catalytic domain of glucoamylases G1 and G2 from Aspergillus niger is produced in vitro in high yield by limited proteolysis using eithe r subtilisin Novo or subtilisin Carlsberg. Purification by affinity ch romatography on an acarbose-Sepharose column followed by ion-exchange chromatography on HiLoad Q-Sepharose leads to separation of a number o f structurally closely related forms of domain. The cleavage occurs pr imarily between Val-470 and Ala-471 as indicated by C-terminal sequenc ing, whereas the N-terminus is intact. Subtilisin Carlsberg, in additi on, produces a type of domain which is hydrolysed before Ser-444, an 0 -glycosylated residue. This leaves the fragment Ser-444-Val-470 disulp hide-bonded to the large N-terminal part of the catalytic domain. Subt ilisin Novo, in contrast, tends to yield a minor fraction of forms ext ending approx. 30-40 amino-acid residues beyond Val-470. The thermosta bility is essentially the same for the single-chain catalytic domain a nd the original glucoamylases G1 and G2, whereas the catalytic domain cut between Ser-443 and Ser-444 is less thermostable. For both types o f domain the kinetic parameters, K(m) and k(cat.), for hydrolysis of m altose are very close to the values found for glucoamylases G1 and G2.