Application of Immobilization Method to Mechanistic Study and Continuous Procedure of Protein Refolding

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(1) In order to select protein refolding media, two different types of immobilized subtilisin BPN' were prepared. One was immobilized on agarose beads by the amino groups of the enzyme and the other was immobilized on agarose beads by the carboxyl groups. Using the quantitatively renaturing preparations obtained by repeated denaturation/renaturation treatment of the immobilized subtilisin, it was revealed that both of two immobilized preparations were useful for media selection.(2) By use of the quantitatively renaturing preparations of subtilisin and thermolysin, organic acids such as 2 M potassium acetate and potassium propionate were found to be excellent media. These solvents were not at all effective to the refolding of hen egg-white Lysozyme.(3) In the case of Streptomyces griseus trypsin (SGT) that involves 3 disulfide bonds in a molecule, the quantitatively renaturing preparation could not be obtained with agarose beads probably because the denaturation/renaturation treatment involved the redox reaction. Instead, immobilization of SGT on glass beads brought about the satisfactory result.(4) In the presence of immobilized benzamidine that represents inhibitory effect to SGT, the refolding yield of SGT in solution system was considerably increased.(5) This fact made it possible to design a continuous and a large-scale refolding of SGT by use of the immobilized inhibitor.