Catalytically active inclusion bodies of Bacillus clausii laccase protein recombinantly produced in E. coli for dye decolorization.

Abstract

Introduction: Pollution causes disease and premature death globally. Various strategies, including biological methods like enzymes, are used to treat pollutants. The production of recombinant proteins in E. coli often results in their aggregation as inclusion bodies (IBs), enriched with the protein of interest. We developed a novel strategy for producing an immobilized bacterial laccase through self-assembly into catalytically active IBs (CatIBs) in E. coli. Our target was the outer spore-coat protein CotA from Bacillus clausii, a halotolerant and pH-stable laccase suitable for scalable applications in wastewater treatment. Two constructs were designed: Lac-17 (without fusion tags) and Lac-40EL (fused to a synthetic 35-amino-acid peptide, named as 40EL), both expressed in E. coli BL21(DE3).

Results: Both strains showed similar growth patterns, though Lac-17 yielded more total recombinant protein (~ 163 mg/L vs. ~127 mg/L). However, Lac-40EL formed CatIBs with up to 22-fold higher enzymatic activity than Lac-17, despite ~ 85% of the recombinant protein being in the insoluble fraction in both cases. Structural models suggest that the 40EL peptide forms an exposed double α-helix that promotes ordered aggregation without hindering the active site of the enzyme. Unlike the soluble fractions, the CatIBs retained their activity after three months at 4 °C. In azo dye decolorization assays with Eriochrome Black T and Congo Red, Lac-40EL CatIBs outperformed Lac-17, particularly in the presence of redox mediators.

Conclusions: This work shows that rational peptide design can improve enzyme immobilization, activity, and stability through CatIBs formation. The resulting CatIBs self-assembled efficiently, were easily recovered, and demonstrated operational stability, supporting their potential for scalable production and novel applications in textile wastewater treatment.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12934-026-02949-4.

Keywords: Auto-immobilized; Contaminants; Dyes; Laccase; Nanoparticles.