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Thermal and Molecular Characterization of Aspergillus awamori Glucoamylase Catalytic and Starch‐Binding Domains
Author(s) -
Khan Saber M. A.,
Reilly Peter J.,
Ford Clark
Publication year - 2000
Publication title -
starch ‐ stärke
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.62
H-Index - 82
eISSN - 1521-379X
pISSN - 0038-9056
DOI - 10.1002/1521-379x(200011)52:11<385::aid-star385>3.0.co;2-g
Subject(s) - aspergillus awamori , chemistry , crystallography , starch , differential scanning calorimetry , circular dichroism , catalysis , thermostability , biochemistry , fermentation , enzyme , thermodynamics , physics
Aspergillus awamori glucoamylase catalytic domain, linker, and starch‐binding domain, the first and third expressed from yeast, have molecular masses of 56.2, 12.6 and 12.9 kDa, respectively, as determined by MALDI‐TOF mass spectroscopy, and have 10.2, 73.2 and 7.0 % (w/w) carbohydrate, respectively, showing overglycosylation by yeast. Unfolding of the starch‐binding domain monitored by circular dichroism is reversible at pH 6.0—8.0, with the unfolding T m and Δ H increasing from 49.7 to 58.5 °C and from 284 to 351 kJ/mol, respectively, as pH decreases from 8.0 to 6.0. The catalytic domain unfolds irreversibly at pH 7.5, producing a single asymmetric endotherm by differential scanning calorimetry, with T m and Δ H at a 1 °C/min heating rate being 60.9 °C and 1720 kJ/mol, but with both increasing as the heating rate increases. This suggests that unfolding is partially under kinetic control, while various tests show that it does not follow a simple two‐state irreversible model. Values of ΔH from calculated solvent‐accessible surface areas of unglycosylated catalytic and starch‐binding domains are about 100 kJ/mol lower than experimentally determined ΔH values of the corresponding glycosylated domains, showing the effect of glycosylation on unfolding enthalpies.