| The unfolding and refolding of Bacillus amyloliquefaciensα-amylase induced with urea or guanidine hydrochloride were studied by using its intrinsic fluorescence emission spectroscopy,fluorescence phase diagram,fluorescence probe,fluorescence quenching, Native-PAGE,SDS-PAGE and Size exclusion chromatography.This study will be helpful to understand how Bacillus amyloliquefaciensα-amylase folded.The results of intrinsic fluorescence emission spectroscopy and fluorescence phase diagram showed that in the unfolding and refolding prosesses ofα-amylase induced with urea and guanidine hydrochloride,there existed a partially folded intermediate,which followed a three-state model.When the guanidine hydrochloride concentration in denaturation/renaturation solution was about 1.0 mol/L,there existed a partially folded intermediate of Bacillus amyloliquefaciensα-amylase,when the urea concentration in denaturation/renaturation solution was about 4.0 mol/L,there also existed a partially folded intermediate of Bacillus amyloliquefaciensα-amylase.So the unfolding and refolding of Bacillus amyloliquefaciensα-amylase induced with urea or guanidine hydrochloride could be considered as a reverse process.The results 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 amyloliquefaciensα-amylase;with the denaturation concentration increasing,the stable hydrophobic regions disappered.the results of fluorescence quenching using acrylamide and potassium iodide as quenchers showed that using acrylamide as quenchers,with the protein denaturation extent increasing,the number of Trp that can be quenched increased untill all the Trp residues were quenched;Using potassium iodide as quenchers,with the maximum number(8) of tryptophan residues in a partially folded intermediate Bacillus amyloliquefaciensα-amylase molecule could be quenched by potassium iodide;with the denaturation concentration increasing,the number of Trp that can be quenched decreased to 5.the results of their protein electrophoreses and SEC showed that no aggregate or aggregate precipitation of Bacillus amyloliquefaciensα-amylase formed during the whole unfolding/refolding procedure of Bacillus amyloliquefaciensα-amylase induced by guanidine hydrochloride or urea.And finally,the suggested unfolding/refolding procedure of Bacillus amyloliquefaciensα-amylase induced by guanidine hydrochloride or urea were presented:during the unfolding and refolding of Bacillus amyloliquefaciensα-amylase induced with guanidine hydrochloride, when the denaturant cocentration was 1.0 mol/L,there exsisted a partially folded intermediate, which followed a three-state model.And no aggregate or aggregate precipitation of Bacillus amyloliquefaciensα-amylase formed during the whole unfolding/refolding procedure of Bacillus amyloliquefaciensα-amylase induced by guanidine hydrochlorideDuring the unfolding and refolding of Bacillus amyloliquefaciensα-amylase induced with urea,when the denaturant cocentration was 4.0 mol/L,there exsisted a partially folded intermediate,which followed a three-state model.And no aggregate or aggregate precipitation of Bacillus amyloliquefaciensα-amylase formed during the whole unfolding/refolding procedure of Bacillus amyloliquefaciensα-amylase induced by urea.
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