Immobilization of Thermoplasma acidophilum Glucose Dehydrogenase and Isocitrate Dehydrogenase Through Enzyme-Inorganic Hybrid Nanocrystal Formation

Abstract

The development of green catalysts, specifically biocatalysts, is crucial for building a sustainable society. To enhance the versatility of biocatalysts, the immobilization of enzymes plays a vital role as it improves their recyclability and robustness. As target enzymes to immobilize, glucose dehydrogenases and carboxylases are particularly important among various kinds of enzymes due to their involvement in two significant reactions: regeneration of the reduced form of coenzyme required for various reactions, and carboxylation reactions utilizing CO2 as a substrate, respectively. In this study, we immobilized Thermoplasma acidophilum glucose dehydrogenase (TaGDH) and T. acidophilum isocitrate dehydrogenase (TaIDH) using a previously reported method involving the formation of enzyme-inorganic hybrid nanocrystals, in the course of our continuing study focusing on carboxylation catalyzed by the free form of TaGDH and TaIDH. Subsequently, we investigated the properties of the resulting immobilized enzymes. Our results indicate the successful immobilization of TaGDH and TaIDH through the formation of hybrid nanocrystals utilizing Mn2+. The immobilization process enhanced TaIDH activity, up to 211%, while TaGDH retained 71% of its original activity. Notably, the immobilized TaGDH exhibited higher activity at temperatures exceeding 87 °C than the free TaGDH. Moreover, these immobilized enzymes could be recycled. Finally, we successfully utilized the immobilized enzymes for the carboxylation of 2-ketoglutaric acid under 1 MPa CO2. In conclusion, this study represents the first immobilization of TaGDH and TaIDH using the hybrid nanocrystal forming method. Furthermore, we achieved significant activity enhancement of TaIDH through immobilization and demonstrated the recyclability of the immobilized enzymes.

Substrate Promiscuity of Thermoplasma acidophilum Malic Enzyme for CO2 Fixation Reaction

“…Notably, this pH range differs from the reported optimal pH for the reductive carboxylation reaction by ME from Pseudomonas diminuta IFO 13182 (pH 6.0) 20 and Thermococcus kodakarensis (pH 6.5) 23 as well as the reported Ta GDH activity for NADPH production (pH 6.5). 24 The differences in optimal pH values may be attributed to the likelihood that the actual pH during the reaction in our experiment was lower than the initial pH due to the formation of H 2 CO 3 and subsequent pH reduction. Considering this factor, the optimum pH for carboxylation with Ta ME was set at 7.5.…”