Chaperonin GroE-facilitated refolding of disulfide-bonded and reduced Taka-amylase A from Aspergillus oryzae.

The refolding characteristics of Taka-amylase A (TAA) from Aspergillus oryzae in the presence of the chaperonin GroE were studied in terms of activity and fluorescence. Disulfide-bonded (intact) TAA and non-disulfide-bonded (reduced) TAA were unfolded in guanidine hydrochloride and refolded by dilution into buffer containing GroE. The intermediates of both intact and reduced enzymes were trapped by GroEL in the absence of nucleotide. Upon addition of nucleotides such as ATP, ADP, CTP or UTP, the intermediates were released from GroEL and recovery of activity was detected. In both cases, the refolding yields in the presence of GroEL and ATP were higher than spontaneous recoveries. Fluorescence studies of intrinsic tryptophan and a hydrophobic probe, 8-anilinonaphthalene-1-sulfonate, suggested that the intermediates trapped by GroEL assumed conformations with different hydrophobic properties. The presence of protein disulfide isomerase or reduced and oxidized forms of glutathione in addition to GroE greatly enhanced the refolding reaction of reduced TAA. These findings suggest that GroE has an ability to recognize folding intermediates of TAA protein and facilitate refolding, regardless of the existence or absence of disulfide bonds in the protein.