Sol-gel immobilization of a thermophilic diguanylate cyclase for enzymatic production of cyclic-di-GMP

Efficient enzymatic production of 3′,5′-cyclic diguanylic acid or cyclic-di-GMP (c-di-GMP) by diguanylate cyclases (DGC) is highly desired given the potential use of the cyclic dinucleotide as vaccine adjuvant and antibacterial agent. The immobilization of DGCs for continuous c-di-GMP production is challenging because the dimerization and conformational changes accompanied with the chemical transformation could be impeded by the entrapment of the enzyme. Here we demonstrate that an engineered thermophilic diguanylate cyclase (etDGC) cloned from Thermotoga maritime can be effectively encapsulated in both the methyltrimethoxysilane (MTMOS) and tetramethoxysilane (TMOS) gels. However, only the enzyme immobilized in the MTMOS gel through a gradual gelation process exhibited enzymatic activity. The immobilized enzyme exhibits extended lifetime and catalytic activity in comparison to the free enzyme in solution, suggesting that the sol-gel immobilized etDGC could be used as a stable and convenient reagent for long-term storage and in situ synthesis of c-di-GMP or ³²P-labelled c-di-GMP. The enhanced stability of the immobilized enzyme also demonstrates the feasibility and potential of continuous production of c-di-GMP on a large scale using sol-gel immobilized etDGC.