Induction, purification and characterization of alpha-N-acetylgalactosaminidase from Aspergillus Niger.
A set of filamentous fungi (42 strains) was screened for alpha-N-acetylgalactosaminidase activity, and a series of inducers and different cultivation conditions were tested. Enzyme production by the best producer Aspergillus niger CCIM K2 was optimized and scaled up. alpha-N-Acetylgalactosaminidase was purified to apparent homogeneity by cation exchange chromatography, gel filtration, and chromatofocusing, and basic biochemical data of the enzyme were determined: The native molecular weight was estimated by gel filtration to be approximately 440 kDa, the molecular weight of the subunit was determined to be 76 kDa and the pI = 4.8. The K (M) was 0.73 mmol/l for o-nitrophenyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside (o-NP-alpha-GalNAc), and optimum enzyme activity was achieved at pH 1.8 and 55 degrees C. This alpha-N-acetylgalactosaminidase is a retaining-type glycosidase, and it was N-deglycosylated without any loss of activity.
Weignerová
L
L
Center for Biotransformation and Biocatalysis, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. lenka@wcontact.cz
Filipi
T
T
Manglová
D
D
Kren
V
V
D001234:Aspergillus niger / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D048809:alpha-N-Acetylgalactosaminidase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D005656:Fungal Proteins; D048809:alpha-N-Acetylgalactosaminidase