Utilization of immobilized archaeal chaperonin for enzyme stabilization

CpkA and CpkB are thermostable chaperonins from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. CpkA or CpkB was immobilized onto agarose beads by the carbodiimide coupling method. In order to investigate whether immobilized CpkA or CpkB stabilizes a foreign enzyme, their ability to stab ilize enzyme was examined using beta -D-galactosidase from Escherichia coli as a model target. in the absence of chaperonin beads, 38.1% of the total soluble protein was precipitated by heat treatment at 53 degreesC for 30 mi n. The protein structure of the residual soluble fraction was examined by c ircular dichroism, indicating that Bo-galactosidase exists as a mixture of the active folded form and the inactive unfolded form. The specific activit y of the residual soluble fraction decreased to 85.1% that of the unheated level (from 149.0 U/mg to 127.0 U/mg), indicating that the active folded fo rm in the heated soluble fraction was 85.2%. In the presence of CpkA- or Cp kB-beads, 28.6% or 34.3% of Bo-galactosidase was precipitated by the same h eat treatment. However, the specific activity in the soluble fraction was a lmost maintained (CpkA, from 151.0 U/mg to 140.3 U/mg; CpkB, from 149.0 U/m g to 140.3 U/mg). These results indicated that CpkA- or CpkB-beads are usef ul for keeping the enzyme active.