title: Heat-stable pullulanase from Bacillus acidopullulyticus: characterization and refolding after guanidinium chloride-induced unfolding.
abstract: Heat-stable pullulanase from Bacillus acidopullulyticus was characterized with respect to its stability against thermal and chemical denaturation and its reactivation after complete chemical unfolding. The enzyme was quite thermostable and retained 55% of activity after heating at 60 degrees C for 30 min at pH 5.5. At pH 6.0, only 9% residual activity was observed. The addition of sucrose, polyols, and Na2SO4 strongly stabilized the enzyme against thermal inactivation. The processes of chemical unfolding by guanidinium chloride (GdmCl) and refolding were studied by enzymological and spectroscopic criteria. B. acidopullulyticus pullulanase was very sensitive to GdmC1 denaturation and had a transition midpoint at 1.2M GdmCl. Reactivation after complete unfolding in 5 M GdmCl was initiated by dilution of the unfolding mixture: 67% reactivation was observed under standard conditions. The influence of some chemical and physical parameters (pH, chemical agents, temperature, and unfolding and refolding time) on refolding was investigated. Of the additives tested to assist reactivation, only bovine serum albumin (BSA) increased the yield of activity to 80%. The full regain of structure and activity was proven by comparing the enzymological, physicochemical, and spectroscopic properties of the native and refolded pullulanase.
authors.affiliation: Institute of Microbiology, Department of Extremophilic Bacteria, Bulgarian Academy of Sciences, Sofia.
authors.forename: M E
authors.initials: ME
authors.lastname: Stefanova
authors.forename: R
authors.initials: R
authors.lastname: Schwerdtfeger
authors.forename: G
authors.initials: G
authors.lastname: Antranikian
authors.forename: R
authors.initials: R
authors.lastname: Scandurra
mesh_terms: D000818:Animals; D001407:Bacillus / Q000201:enzymology*; D002417:Cattle; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D019791:Guanidine; D006361:Heating; D017510:Protein Folding
chemical_list: D006026:Glycoside Hydrolases; C100181:pullulanase; D019791:Guanidine