title: The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis.
abstract: 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.
authors.affiliation: School of Food Biosciences, The University of Reading, PO Box 226, Whiteknights, Reading RG6 6 AP, UK.
authors.forename: Vasileios I
authors.initials: VI
authors.lastname: Athanasopoulos
authors.forename: Keshavan
authors.initials: K
authors.lastname: Niranjan
authors.forename: Robert A
authors.initials: RA
authors.lastname: Rastall
mesh_terms: 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*
chemical_list: D007527:Isoenzymes; D009844:Oligosaccharides; D008345:Manganese; D043323:alpha-Mannosidase