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The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

Athanasopoulos, V.I., Niranjan, K. and Rastall, R.A. (2005) The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis. Carbohydrate Research, 340 (4). pp. 609-617. ISSN 0008-6215

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To link to this article DOI: 10.1016/j.carres.2005.01.005

Abstract/Summary

1,6-alpha-D-Mannosidase from Aspergillits 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 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-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocus sing 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 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. (c) 2005 Elsevier Ltd. All rights reserved.

Item Type:Article
Refereed:Yes
Divisions:Faculty of Life Sciences > School of Chemistry, Food and Pharmacy > Department of Food and Nutritional Sciences
ID Code:13571
Uncontrolled Keywords:1,6-alpha-D-mannosidase, enzyme purification, linkage specificity, glycosidase, REGIOSELECTIVE SYNTHESIS, PENICILLIUM-CITRINUM, GEL ELECTROPHORESIS, YEAST MANNAN, GLYCOSIDASES, 1,2-ALPHA-D-MANNOSIDASE, NIGER, 1,2-ALPHA-MANNOSIDASE, SAITOI, OLIGOSACCHARIDES

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