The production, purification and characterisation of two novel α-d-mannosidases from Aspergillus phoenicis Original Research Article

1,6-α-d-Mannosidase from Aspergillus phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-α-d-Mannosidase had a temperature optimum of 60 °C, a pH optimum of 4.0–4.5, a Km of 14 mM with α-d-Manp-(1→6)-d-Manp as substrate. It was strongly inhibited by Mn2+ and did not need Ca2+ or any other metal cofactor of those tested. The enzyme cleaves specifically (1→6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-α-d-mannopyranoside or baker’s yeast mannan. 1,3(1,6)-α-d-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-α-d-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves α-d-Manp-(1→3)-d-Manp 10 times faster than α-d-Manp-(1→6)-d-Manp, has very low activity towards p-nitrophenyl-α-d-mannopyranoside and baker’s yeast mannan, and no activity towards α-d-Manp-(1→2)-d-Manp. The activity towards (1→3)-linked mannobiose is strongly activated by 1 mM Ca2+ and inhibited by 10 mM EDTA, while (1→6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities.