The Effects Of Urea, Guanidine Hydrochloride, Ca²⁺ And Cl^- On The Molecule Structure Of Porcine Pancreatic And Bacillus Amyloliquefaciens α-amylases

This thesis is composed of two technical parts.In the first part, we study the distribution and transition of each stable conformational state ofα-amylase molecules under different urea and guanidine hydrochloride concentrations in denaturation solution. The key step is to acquire two characteristic unfolding parameters K and m, through measuring the residual activity ratios ofα-amylase under different urea and guanidine hydrochloride concentrations in denaturation solution. According to the measurement, in the process of unfolding of Bacillus amyloliquefaciensα-amylase induced by guanidine hydrochloride, the characteristic unfolding parameters m1, m2, K1, and K2 are 5.36×10-1,6.42×10-1,2.09 and 0.35, respectively; in its unfolding process induced by urea, the parameters m1, m2, K1 and K2 are 1.52,1.77,4.01×10-1 and 8×10-3, respectively; in the process of unfolding of Porcine pancreaticα-amylase induced by guanidine hydrochloride, the parameters m1, m2, m3, K1, K2 and K3 are 0.94,1.52,4.85,3.16×10-1,3.48 and 1.36×10-4, respectively; and in its unfolding process induced by urea, m1, m2, K1 and K2 are 4.50×10-1,1.63,1.24 and 4.98×10-2, respectively. The results show that during the unfolding of Bacillus amyloliquefaciensα-amylase molecules and Porcine pancreaticα-amylase molecules induced by urea, the unfolding of Porcine pancreaticα-amylase molecules is easier than the unfolding of Bacillus amyloliquefaciensα-amylase molecules; while during the unfolding of Bacillus amyloliquefaciensα-amylase molecules and Porcine pancreaticα-amylase molecules induced by guanidine hydrochloride, the unfolding of Bacillus amyloliquefaciensα-amylase molecules is easier than the unfolding of Porcine pancreaticα-amylase molecules.In the second part, we study the change of secondary structure and tertiary structure of Bacillus amyloliquefaciensα-amylase under different Ca2+ and Cl- concentrations, using fluorescence inspire spectrometry, fluorescence probe techniques, fluorescence quenching techniques and Fourier transform infrared spectroscopy. The results of fluorescence inspire spectrometry, fluorescence probe techniques and fluorescence quenching techniques show that the tertiary structure of Bacillus amyloliquefaciensα-amylase remains the same under different Ca2+ and Cl- concentrations. The results of Fourier transform infrared spectroscopy show that with a gradual increase in the Ca2+ concentration, theα-Helix content in the Bacillus amyloliquefaciensα-amylase increases from 5.82% to 30.14%, the disorder content decreases from 45.23% to 19.08%, and there is no obvious change in theβ-sheet orβ-turn content; while with a gradual increase in the Cl- concentration, theα-Helix content in the Bacillus amyloliquefaciensα-amylase increases from 5.82% to 10.24%, the disorder content decreases from 45.23% to 38.34%, and there is no obvious change in theβ-sheet orβ-turn content either.