[P003] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P02-255] Recovery of an Active Hyperthermophilic Arginine Decarboxylase by High-Temperature Refolding

○Daigo Fujisaki¹, Yuri Ishii¹, Shinsuke Fujiwara¹ (1. Kwansei-Gakuin University (Japan))

Keywords:

Agmatine,Arginine decarboxylase,Hyperthermophilic enzyme,Pyrobaculum calidifontis,Protein refolding,Thermostable biocatalyst

Agmatine is a bioactive polyamine produced through the decarboxylation of the basic amino acid L-arginine. Recent studies suggest that agmatine may contribute to reducing the risk of dementia and other neurological disorders, making it an increasingly important compound in aging societies (1). Agmatine is also marketed as a muscle-enhancing supplement in sports facilities and health stores; however, its relatively high production cost limits broader applications. Agmatine can be synthesized enzymatically from L-arginine by arginine decarboxylase. Because the solubility of arginine increases at elevated temperatures, high-temperature reactions allow higher substrate concentrations and may improve production efficiency. Thermostable enzymes are therefore attractive catalysts for this process. In this study, we aimed to obtain an active form of the hyperthermophilic arginine decarboxylase from Pyrobaculum calidifontis and evaluate conditions required for its proper folding and stability. The gene encoding arginine decarboxylase (Pc-speA) was cloned and heterologously expressed in Escherichia coli. However, recombinant Pc-SpeA predominantly formed insoluble aggregates. These aggregates were collected and solubilized using guanidine hydrochloride, and the unfolded protein was subjected to refolding by dialysis. Refolding at cold and moderate temperatures failed to recover enzymatic activity. Interestingly, when the refolding process was performed at elevated temperatures (40–80 °C), active soluble Pc-SpeA was obtained only when refolding was conducted above 60 °C. This result suggests that proper folding of this hyperthermophilic enzyme requires high-temperature conditions consistent with its native environment (2). The refolded enzyme maintained structural integrity and catalytic activity when stored at elevated temperatures, whereas storage at low temperatures (0–20 °C) caused a marked loss of activity. These findings demonstrate the effectiveness of high-temperature refolding for recovering active hyperthermophilic enzymes while also revealing a practical limitation in low-temperature stability. To address this issue, we are currently investigating compatible solutes and other stabilizing agents to maintain enzyme activity during low-temperature storage.

(1) Akasaka, N. and Fujiwara, S. Amino acids 52, 181-197 (2020)
(2) Maekawa, K. et al., Biochem. Biophys. Res. Commun. 786, 152705 (2025)