title: Effect of glycosylation of bacterial amylase on stability and active site conformation.
abstract: With a view to understand the changes in the conformation of bacterial amylase, the enzyme preparation was conjugated to dextran. Glycosylation of purified bacterial amylase resulted in increased stability against heat, proteolytic enzymes and denaturing agents. Several group specific inhibitors exhibited dose-dependent inhibition and the extent of inhibition was same for native as well as for the glycosylated enzyme. The pH optima of native and glycosylated enzyme remained the same indicating that the ionization at the active site is not greatly influenced as a result of glycosylation. Although the native as well as the glycosylated enzyme bind to the substrate with the same affinity, the rate of reaction differed greatly at 90 and 100 degrees C. At 70 degrees C, the rate of reaction was similar for the conjugated as well as the unconjugated amylase. Thermostability at different temperatures clearly showed that the glycosylated enzyme had greater stability compared to the native enzyme. The divalent cation binding site in the amylase also appears to be unaltered upon glycosylation since EDTA inhibited both enzymes to the same extent and addition of calcium ion restored the activity to almost the same level. These studies showed that conjugating the amylase enzyme with a bulky molecule like dextran does not affect the conformation at the active site.
authors.affiliation: Biochemistry Division, Regional Research Laboratory, Jorhat, Assam, India.
authors.forename: R A
authors.initials: RA
authors.lastname: Srivastava
mesh_terms: D000681:Amylases / Q000378:metabolism*; D001665:Binding Sites; D003911:Dextrans / Q000378:metabolism*; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006031:Glycosylation; D007700:Kinetics; D011487:Protein Conformation; D000516:alpha-Amylases / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D003911:Dextrans; C070530:amylase-dextran conjugate; D000681:Amylases; D000516:alpha-Amylases