Int J Biol Macromol. 2026 Apr 12:151983. doi: 10.1016/j.ijbiomac.2026.151983. Online ahead of print.

ABSTRACT

4,5-DOPA extradiol dioxygenase (DODA) is a key enzyme in the catabolism of aromatic compounds and holds significant promise for biocatalysis applications. This study presents the first successful immobilization of recombinant DODA with a C-terminal His-tag, paving the way for in-vitro biocatalytic systems using this enzyme. While the N-terminal His-tagged DODA was unable to withstand immobilization, losing 78% of its activity, the C-terminal His-tagged DODA, expressed in inclusion body, was efficiently refolded, purified, and stabilized by immobilization. The activity of the refolded enzyme was markedly increased by the presence of divalent metal ions, especially Ca²⁺. In addition, various surfactants and organic solvents had different effects on the enzyme functionality. The C-terminal His-tagged DODA was covalently immobilized onto epoxy-functionalized silica, achieving an 80% immobilization yield and almost a tenfold increase in specific activity compared to the free enzyme. Immobilization shifted the enzyme’s optimal temperature from 37 °C to 50 °C, while its optimal pH remained at 8.5. The immobilized enzyme demonstrated improved thermal stability, retaining 38% of its activity at 60 °C and showing increased resistance to thermal inactivation. Its storage stability was enhanced after immobilization by retaining 50% of its original activity after eight weeks at 4 °C, whereas the free enzyme lost activity within one week under the same condition. Immobilized DODA also retained 76% activity after seven cycles, showing high reusability.

PMID:41980693 | DOI:10.1016/j.ijbiomac.2026.151983