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 stabilize 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 degrees C for 30 min. The protein structure of the residual soluble fraction was examined by circular dichroism, indicating that beta-D-galactosidase exists as a mixture of the active folded form and the inactive unfolded form. The specific activity 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 form in the heated soluble fraction was 85.2%. In the presence of CpkA- or CpkB-beads, 28.6% or 34.3% of beta-d-galactosidase was precipitated by the same heat treatment. However, the specific activity in the soluble fraction was almost maintained (CpkA, from 151.0 U/mg to 140.3 U/mg; CpkB, from 149.0 U/mg to 140.3 U/mg). These results indicated that CpkA- or CpkB-beads are useful for keeping the enzyme active.