Expression in Aspergillus niger of the starch-binding domain of glucoamylase. Comparison with the proteolytically produced starch-binding domain.
Glucoamylase 1 from Aspergillus niger is an economically important enzyme in many industrial processes. It hydrolyses granular starch and comprises two distinct domains, a catalytic and a starch-binding domain (SBD). We have transformed A. niger with an expression vector for the secretion of SBD for physico-chemical studies. This was achieved by introducing into the glucoamylase gene a short sequence encoding an endoproteolytic cleavage recognition site such that free SBD was secreted at yields up to 200 mg/l. Free SBD was also obtained by proteolytic digestion of full-length glucoamylase 1. Electrospray mass spectroscopy was used to determine the carbohydrate content of both SBDs. It revealed that the engineered one is more glycosylated: an average of three mannose residues compared to one for the proteolytically derived SBD. Sequencing results also suggest partial glycosylation for the three Thr residues involved (510, 511, 513). It is probable that the engineered SBD represents the true glycosylation level of the SBD in native glucoamylase. Binding of beta-cyclodextrin to the SBD was investigated. It was found that the stoichiometry and the spectral perturbation of Trp residues were identical for both SBDs, but the engineered SBD bound less strongly to the ligand. At high concentrations of beta-cyclodextrin relative to the estimated Kd values, the maximum absorbance changes were identical. The observed difference at low beta-cyclodextrin levels was probably due to the higher level of glycosylation of the expressed SBD. We conclude that the proteolytically derived and expressed starch binding domains both bind 2 mol beta-cyclodextrin/mol protein, but that the pattern of glycosylation and strength of binding are different.
Le Gal-Coëffet
M F
MF
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Jacks
A J
AJ
Sorimachi
K
K
Williamson
M P
MP
Williamson
G
G
Archer
D B
DB
D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003505:Cyclodextrins / Q000378:metabolism; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D013213:Starch / Q000378:metabolism*; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase; C031215:betadex