REFOLDING OF SUBTILISIN BPN ACHIEVED ALMOST QUANTITATIVELY BY COVALENT IMMOBILIZATION ON AN AGAROSE-GEL

Renaturation of subtilisin BPN' could successfully be attained in the case of subtilisin immobilized covalently on an agarose gel in the med ium containing 2 M potassium acetate or other salts. The maximum yield of renaturation based on enzymatic activity was as high as 80% in the presence of 2 M potassium acetate from the fully denatured immobilize d subtilisin. Preliminary denaturation in 6 M guanidine hydrochloride at pH 2.4 for 4 h rendered 20-30% of immobilized subtilisin to release from the agarose gel, which was estimated by reverse-phase high perfo rmance liquid chromatography as well as by electrophoresis. We conclud ed that, in the presence of 2 M potassium acetate, we could achieve al most quantitative refolding of subtilisin by immobilization. Next, the rate of renaturation of denatured immobilized subtilisin was compared among several media containing several salts at the concentration of 2 M, at 25-degrees-C. The times for 50% renaturation, t1/2S, in the pr esence of potassium acetate, potassium chloride, and lithium chloride were about 24, 63, and 153 min with apparent ultimate yields of 80, 71 , and 40%, respectively. Higher rates and yields resulted also in the presence of the other organic salts such as dipotassium succinate and potassium propionate.