The Immobilization of a Cyclodipeptide Synthase Enables Biocatalysis for Cyclodipeptide Production.

Cyclodipeptide synthases (CDPSs) are enzymes that use aminoacylated tRNAs as substrates to produce cyclic dipeptide natural products acting as anticancer and neuroprotective compounds. Many CDPSs, however, suffer from instability and poor recyclability, while enzyme immobilization can enhance catalyst efficiency and reuse. Here, the CDPS enzyme from Parcubacteria bacterium RAAC4_OD1_1 was immobilized using three different supports: biochar from waste materials, calcium-alginate beads, and chitosan beads. Immobilization of active PbCDPS was successful, and production of the cyclodipeptide cyclo (His-Glu) (cHE) was confirmed by HPLC-MS. Biochar from spent coffee activated with glutaraldehyde, alginate beads, and chitosan beads activated with glutaraldehyde led to a 5-fold improvement in cHE production, with the immobilized enzyme remaining active for seven consecutive cycles. Furthermore, we co-immobilized three enzymes participating in the cascade reaction yielding cHE (PbCDPS, histidyl-tRNA synthetase, and glutamyl-tRNA synthetase). The enzymatic cascade successfully produced the cyclic dipeptide, underscoring the potential of immobilizing various enzymes within a single support. Importantly, we demonstrated that tRNAs remained free in solution and were not adsorbed by the beads. We paved the way for the immobilization of enzymes that utilize tRNAs and other complex substrates, thereby expanding the range of reactions that can be exploited by using this technology.