The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis.
1,6-alpha-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-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5, a K(m) of 14 mM with alpha-D-Manp-(1-->6)-D-Manp as substrate. It was strongly inhibited by Mn(2+) and did not need Ca(2+) 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-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-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)-alpha-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 alpha-D-Manp-(1-->3)-D-Manp 10 times faster than alpha-D-Manp-(1-->6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1-->2)-D-Manp. The activity towards (1-->3)-linked mannobiose is strongly activated by 1mM Ca(2+) and inhibited by 10mM 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.
Athanasopoulos
Vasileios I
VI
School of Food Biosciences, The University of Reading, PO Box 226, Whiteknights, Reading RG6 6 AP, UK.
Niranjan
Keshavan
K
Rastall
Robert A
RA
D001230:Aspergillus / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007527:Isoenzymes; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification*; D013379:Substrate Specificity; D043323:alpha-Mannosidase / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification*
D007527:Isoenzymes; D009844:Oligosaccharides; D008345:Manganese; D043323:alpha-Mannosidase