title: Media selection for refolding of thermolysin by use of immobilized preparation.
abstract: Selection of the most effective medium for the correct refolding of thermolysin was performed. Thermolysin that had been denatured with 6 M guanidinium chloride at pH 2.0 could not be recovered to its activity larger than ca. 10% even when the denaturant was diluted with a conventional buffer solution. The amount of activity recovered by this method decreased with time. The recovered activity was ca. 20% at most where 1 M calcium chloride or 1.6 M calcium acetate was employed as the refolding medium instead of the conventional buffer solution. In this case also, the activity decreased with time. Not only the low recovered activity or yield, but also the elimination of the activity once recovered, was probably mainly due to the intermolecular interactions between protein molecules such as autolysis and aggregation. In order to exclude the influence of the intermolecular interactions and to select the effective media for the correct refolding of thermolysin, immobilized thermolysin was prepared using agarose gel. Employment of the immobilized preparation made it possible to quantitatively determine the refolding of thermolysin and results revealed that the salts of organic acid, such as potassium acetate and sodium acetate, were excellent media for refolding. The immobilization was confirmed to be available for the selection of protein refolding media and indispensable, especially in the case of proteases. Since these results were partly similar to those obtained in the case of subtilisin reported previously, results of both cases were compared.
authors.affiliation: Department of Chemical Reaction Engineering, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors.forename: D
authors.initials: D
authors.lastname: Nohara
authors.forename: Y
authors.initials: Y
authors.lastname: Senga
authors.forename: M
authors.initials: M
authors.lastname: Matsubara
authors.forename: T
authors.initials: T
authors.lastname: Sakai