OPERATION CONDITIONS OF ENZYME REFOLDING BY CHAPERONIN AND RECYCLE SYSTEM USING ULTRAFILTRATION

To clarify the efficient refolding conditions of enzymes using chapero nin GroE from Escherichia coli, the effects of various factors on the chaperonin-mediated refolding of enzymes from 4M guanidine hydrochlori de (GdnHCl) were investigated. Three enzymes, Bacillus subtilis alpha- amylase, bovine deoxyribonuclease I (DNase I) and yeast enolase were u sed as the model systems. In all enzymes, the maximum recovery of acti vities (90-150% with respect to the enzyme activities before denaturat ion) was attained in the presence of 2mM ATP and 2-5-fold molar excess of GroE 21-mer or GroEL 14-mer over enzyme molecules. Since the recov ery of enzyme activity by GroEL is close to that by GroE, GroEL as wel l as GroE are applicable for the refolding systems of these enzymes. G roE significantly enhanced the recovery of enzyme activities at around 25-40 degrees C, pH 6-9 and up to rather high final GdnHCl and enzyme concentrations. Therefore, the chaperonin efficiently mediates the en zyme refolding under wide operation conditions. To test the reusabilit y of chaperonins in enzyme refolding, GroEL was separated from refolde d enzymes by ultrafiltration and recycled. GroEL repeatedly mediated t he refolding of enzymes by choosing appropriate membranes. Therefore, protein refolding processes based on chaperonins are promising. (C) 19 97 Elsevier Science S.A.