ABSTRACT
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.
Subject(s)
Aspergillus niger/enzymology , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , alpha-N-Acetylgalactosaminidase/chemistry , alpha-N-Acetylgalactosaminidase/isolation & purification , Aspergillus niger/metabolism , Enzyme Activation , Enzyme Stability , Fungal Proteins/metabolism , Kinetics , Molecular Weight , alpha-N-Acetylgalactosaminidase/metabolismABSTRACT
4-Nitrophenyl alpha-D-galactopyranosyl-(1-->3)-6-O-acetyl-alpha-D-galactopyranoside was prepared in a transglycosylation reaction catalyzed by alpha-D-galactosidase from Talaromyces flavus using 4-nitrophenyl alpha-D-galactopyranoside as a glycosyl donor and 4-nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside as an acceptor. 4-Nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside and 4-nitrophenyl 6-O-acetyl-beta-D-galactopyranoside were prepared in a regioselective enzymic transesterification in pyridine-acetone catalyzed by the lipase PS from Burkholderia cepacia. A series of water-miscible organic solvents (acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, 2-methoxyethanol, pyridine, 2-methylpropan-2-ol, tetrahydrofuran, propargyl alcohol) were used as co-solvents in this enzymic reaction. Their influence on the activity and stability of the alpha-galactosidase from T. flavus was established. 2-Methylpropan-2-ol and acetone (increasing the solubility of the modified substrate acceptors and displaying the minimum impairment of the activity and stability of the enzyme) were used as co-solvents in transglycosylation reactions.
Subject(s)
Disaccharides/chemical synthesis , Nitrophenylgalactosides/chemical synthesis , alpha-Galactosidase/chemistry , Burkholderia cepacia/enzymology , Carbohydrate Conformation , Carbohydrate Sequence , Catalysis , Disaccharides/biosynthesis , Magnetic Resonance Spectroscopy , Molecular Structure , Nitrophenylgalactosides/biosynthesis , Solvents , Talaromyces/enzymology , alpha-Galactosidase/metabolismABSTRACT
Lincomycin (1), a glycosidic antibiotic, active against Gram-positive bacteria, was modified enzymatically with the aim of improving its physico-chemical and biological properties. Compound 1 was glycosylated using jack bean alpha-mannosidase to produce 7-O-alpha-D-mannopyranosyl-lincomycin (2).
