UBC Theses and Dissertations
UBC Theses and Dissertations
Purification and characterization of alpha-amylase from Bacteroides amylophilus strain H-18 Rahman, Sheikh Saif-Ur
The research was undertaken to study the extracellular α-amylase produced by the anaerobic rumen bacterium, Bacteroides amylophilus strain H-18. Four active isoenzymes of α-amylase were detected by disc electrophoresis and electrofocusing techniques. Isoelectric points as determined by electrofocusing were pH 3.7, 4.5, 5.9 and 8.0. Isoenzymes were named 1, 2, 3 and 4 with respect to their increasing isoelectric points. α-Amylase isoenzyme 1 was purified by DEAE-Sephadex and G-200 techniques. Some of its general physio-chemical properties were studied. It had maximum activity at pH 6.7, 44°C and was stabilized by calcium ion. It was susceptible to thermal denaturation in the absence of calcium. Various other metal ions tested could not replace the calcium in regenerating maximum activity. It was found by atomic absorption spectrophotometry that α-amylase isoenzyme 1 contained 3 gram-atoms of calcium per mole of enzyme. The estimated molecular weight by gel filtration technique was 45,000 Daltons. Amino acid analysis indicated the absence of cysteine, therefore, disulphide linkages were not involved in maintaining the tertiary structure. Tryptophan appeared to be required for enzymic activity, as determined by the N-bromosuccinamide oxidation technique. The mode of action of α-amylase isoenzyme 1 was studied using amylose and soluble starch as substrates. The products of enzymatic degradation were analysed qualitatively by thin layer chromatography. The maltohexaose, maltoheptaose, maltoctaose, maltonanaose and maltodecaose remained in the digest mixture for sometime after the achroic point. The degree of multiple attack was 2, as calculated by determining the ratio of the reducing value of the oligosaccharide fraction to that of polysaccharide fraction. Antisera against α-amylase isoenzyme 1, produced in rabbits by injection of α-amylase in Freund's complete adjuvant was found to be mono-specific. The inhibition of α-amylase activity by antibody and inhibitory effect of starch on the amylase-anti-amylase system were demonstrated. The effect of anti-amylase (isoenzyme 1) globulin on amylases of diverse origin was studied by the Ouchterlony double diffusion technique. These experiments demonstrated antigenic determinants which were distinct from those present on the α-amylase of hog pancreas, Bacillus subtilis and Aspergillus oryzae. Immunoelectrophoretic analysis indicated the presence of only a single antigenic component. Quantitative precipitation studies gave a typical curve with one equivalence point with an antibody to antigen ratio of 2.31. N-Bromosuccinimide treated α-amylase (isoenzyme 1) exhibited similar immunochemical behaviour to the native enzyme, but completely lost its catalytic activity. It is possible that catalytic and antigenic sites were distinct. Urea treated α-amylase (isoenzyme 1) did not show any precipitate with its specific antibody and thus appeared to have lost its antigenic structure.
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