Catalytic properties of Sepharose-bound L-alanine dehydrogenase from Bacillus cereus.
(1) L-Alanine dehydrogenase from Bacillus cereus was purified by a two-step chromatographic procedure involving Cibacron-Blue 3G-A Sepharose 4B-CL, and Sepharose 6B-CL, and immobilized on CNBr-activated Sepharose 4B. (2) Following immobilization via two of the six subunits, L-alanine dehydrogenase retained 66% of the specific activity of the soluble enzyme. The affinity of the immobilized enzyme for NH4+, pyruvate and L-alanine, was not different to that of the soluble form. The Km of the Sepharose-bound L-alanine dehydrogenase for pyridine coenzymes was 6-8-times higher than in the soluble case. (3) The stability of L-alanine dehydrogenase towards urea or thermal denaturation was increased by immobilization. (4) The incubation at 37 degrees C for 24 h of the immobilized L-alanine dehydrogenase with 3 M NH4Cl/NH4OH buffer (pH 9) released 70% of the enzyme. The specific activity and the affinity of the 'solubilized' L-alanine dehydrogenase for the pyridine coenzymes was the same as that obtained with the original, soluble L-alanine dehydrogenase.
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D004800:Enzymes, Immobilized / Q000378:metabolism; D007700:Kinetics; D011489:Protein Denaturation; D012685:Sepharose; D012995:Solubility; D014508:Urea
D004800:Enzymes, Immobilized; D014508:Urea; D012685:Sepharose; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase