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

## TL;DR

Researchers immobilized a cyclodipeptide synthase enzyme on different supports to improve its stability and reusability for producing beneficial cyclic dipeptides.

## Contribution

The immobilization of three enzymes using tRNA substrates in a cascade reaction for cyclodipeptide production is novel.

## Key findings

- Immobilization on biochar, alginate, and chitosan beads improved cyclodipeptide production by 5-fold.
- The immobilized enzymes remained active for seven consecutive cycles.
- tRNAs were not adsorbed by the beads, enabling their reuse in the reaction.

## Abstract

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 bacteriumRAAC4_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.

Immobilizing three enzymes utilizing
complex aminoacyl-tRNAs
substrates, we enabled a sustainable cascade for green production
of cyclodipeptide natural products.