Effect of lysine modification on the conformational stability of a-Amylase / Tan Cheau Yuaan
Chemical modifications of Bacillus licheniformis �–amylase (BLA) were carried out using 100 molar excess of succinic anhydride over protein, 0.66 M potassium cyanate and 0.46 M O-methylisourea under standard conditions to prepare different modified derivatives. The percentage of modification as d...
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| Format: | Thesis |
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2011
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| Online Access: | http://pendeta.um.edu.my/client/default/search/results?qu=Effect+of+lysine+modification+on+the+conformational+stability&te= http://studentsrepo.um.edu.my/3569/4/TITLE_PAGE%2C_ABSTRACT%2C_CONTENT_PAGE.pdf http://studentsrepo.um.edu.my/3569/5/FULL_CHAPTERS.pdf http://studentsrepo.um.edu.my/3569/6/REFERENCES.pdf |
| Summary: | Chemical modifications of Bacillus licheniformis �–amylase (BLA) were carried out
using 100 molar excess of succinic anhydride over protein, 0.66 M potassium cyanate
and 0.46 M O-methylisourea under standard conditions to prepare different modified
derivatives. The percentage of modification as determined by TNBSA reaction method
was found to be 42% for succinylated (S42), 81% for carbamylated (C81) and 88% for
guanidinated (G88) BLA derivatives. These modified preparations were checked for
their size and charge homogeneity by gel chromatography on Sephacryl S-200 HR
column and polyacrylamide gel electrophoresis. Conformational changes in these
preparations were detected by analytical gel chromatography on a calibrated Sephacryl
S-200 HR column by determining the Stokes radii of native and modified derivatives.
C81 BLA was found to have the highest Stokes radius value followed by S42 and G88
BLAs. Conformational stabilities of native and modified BLA derivatives were
investigated by urea denaturation using circular dichroism (CD) spectroscopy.
Denaturation profiles showed a decrease in the stability of modified BLAs being more
pronounced in C81 BLA. In addition, the effect of calcium on urea and guanidine
hydrochloride (GdnHCl) denaturation of native and calcium-depleted enzymes was also
analyzed by CD, fluorescence spectroscopy and biological activity. Based on these
results, calcium was found to stabilize the enzyme against both urea and GdnHCl
denaturations. |
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