关键词: 淀粉液化芽孢杆菌a-淀粉酶, 去折叠, 折叠中间态, 盐酸胍

The unfolding of Bacillus amyloliquefaciens a-amylase induced by guanidine hydrochloride was studied by using its intrinsic fluorescence spectroscopy, fluorescence phase diagram, fluorescence probe, fluorescence quenching, protein electrophoresis and chromatography. The results of its intrinsic fluorescence spectroscopy and fluorescence phase diagram showed that when the guanidine hydrochloride concentration in denaturation solution was about 1.0 mol/L, there existed a partially folded intermediate of Bacillus amyloliquefaciens a-amylase during its unfolding procedure, which followed a three-state model; the result of its fluorescence probe showed that when the guanidine hydrochloride concentration in denaturation solution was about 1.0 mol/L, there existed some stable hydrophobic regions, which could interact with a hydrophobic reagent 8-anilino-1-naphthalene sulfonic acid (ANS), in the partially folded intermediate of Bacillus amyloliquefaciens a-amylase; and the results of fluorescence quenching using acrylamide and potassium iodide as quenchers showed the distribution of Trp residues in Bacillus amyloliquefaciens a-amylase in different denaturation solution, with the maximum number (8) of tryptophan residues in a partially folded intermediate Bacillus amyloliquefaciens a-amylase molecule could be quenched by potassium iodide; and the results of their protein electrophoresis and SEC showed that no aggregate or aggregate precipitation of Bacillus amyloliquefaciens a-amylase formed during the whole unfolding procedure of Bacillus amyloliquefaciens a-amylase induced by guanidine hydrochloride. And finally, a suggested unfolding procedure of Bacillus amyloliquefaciens a-amylase induced by guanidine hydrochloride was presented.

Key words: Bacillus amyloliquefaciens a-amylase, unfolding procedure, intermediates, guanidine hydrochloride