Date of Award

11-1-2016

Thesis Type

masters

Document Type

Thesis (Restricted Access)

Divisions

science

Department

Faculty of Science

Institution

University of Malaya

Abstract

The influence of buffer composition on the conformational stability of native and calcium-depleted Bacillus licheniformis α-amylase (BLA) was investigated against guanidine hydrochloride (GdnHCl) denaturation using circular dichroism, fluorescence and UV-difference spectroscopy. Buffers used in these experiments were: 0.05 M sodium phosphate buffer, pH 7.5, 0.15 M Tris-HCl buffer, pH 7.5, 0.15 M HEPES buffer, pH 7.5 and 0.15 M MOPS buffer, pH 7.5. Differential effects of buffer composition on GdnHCl denaturation of BLA were evident from the magnitude of these spectral signals, which followed the order: sodium phosphate > Tris-HCl > HEPES > MOPS. These effects became more pronounced when calcium-depleted BLA was used in the incubation mixture as revealed by a lower relative mean residue ellipticity, lower relative fluorescence intensity, and higher change in emission maximum. Depletion of calcium from BLA suggested a decrease in the protein conformational stability. Gel chromatographic analyses of native, 3 M GdnHCldenatured and 6 M GdnHCl-denatured BLAs were made in different runs on Sephacryl S-200 HR column (1.0×30 cm), equilibrated with these buffers. The results obtained clearly suggested formation of similar denatured states and aggregated forms of BLA in 3 M and 6 M GdnHCl in the presence of these buffers. However, quantitative differences in BLA aggregation were noticed in these buffers in the presence of 6 M GdnHCl. In view of the above, spectral results on BLA stability against GdnHCl obtained with different probes (MRE, fluorescence intensity and emission maximum) in different buffers should be treated with caution.

Note

Dissertation (M.A.) – Faculty of Science, University of Malaya, 2016.

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