Immobilization of formate dehydrogenase from Candida boidinii through cross-linked enzyme aggregates

• Formate dehydrogenase from Candida boidinii was immobilized through cross-linked enzyme aggregates (CLEA).
• It was shown that the kinds and concentrations of the cross-linker, the cross-linking time, and additives such as a proteic feeder had significant effects on the activity recovery of CLEAs.
• Residual activity of FDH CLEA prepared under optimal conditions was over 95% after 10 times reuse.
• CLEAs of FDH could be utilized efficiently for both NADH regeneration and CO2 reduction.

We employed a cross-linked enzyme aggregate (CLEA) method to immobilize formate dehydrogenase (FDH) from Candida boidinii. The optimal conditions for the preparation of CLEAs were determined by examining effects of various parameters: the nature and amount of cross-linking reagent, additive concentration, cross-linking time, and pH during CLEA preparation. The recovered activities of CLEAs were significantly dependent on the concentration of glutaraldehyde; however, the recovered activity was not severely influenced by the content of dextran polyaldehyde as a mild cross-linker. Bovine serum albumin (BSA) was also used as a proteic feeder and enhanced the activity recovery by 130%. The highest recovered activity of CLEA was 18% for formate oxidation reaction and 25% for CO2 reduction reaction. The residual activity of CLEA prepared with dextran polyaldehyde (Dex-CLEA) was over 95% after 10 cycles of reuse. The thermal stability of Dex-CLEA was increased by a factor of 3.6 more than that of the free enzyme. CLEAs of FDH could be utilized efficiently for both NADH regeneration and CO2 reduction.

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