Purification of Sulfhydryl Oxidase

Significance of the topic

Enzymes are critical catalysts in modern food production, offering specific and efficient biocatalysis under mild conditions. Sulfhydryl oxidase (SOx), which promotes disulfide bond formation, holds promise as a natural stabilizer in protein-rich foods such as baked goods and egg preparations. Its commercial rarity and broad application potential underscore the importance of developing robust purification strategies.

Objectives and study overview

This work describes the development of an affinity chromatography-based purification protocol for recombinant His-tagged sulfhydryl oxidase. The primary goals were to optimize binding and elution conditions using immobilized metal ion chromatography (IMAC) and to evaluate enzyme purity and yield to support further functional and application testing.

Methodology and instrumentation used

Sulfhydryl oxidase was overexpressed in Bacillus subtilis and recovered from clarified fermentation supernatant. The clarified broth was ultrafiltered, then applied to a Chelating Sepharose Fast Flow column preloaded with Ni²⁺. After washing with buffer A (20 mM Na₃PO₄, 300 mM NaCl, 10 mM imidazole, pH 7.4), the His-tagged enzyme was eluted with buffer B (20 mM Na₃PO₄, 300 mM NaCl, 250 mM imidazole, pH 7.4) on an AZURA® Bio purification system. Protein elution was monitored by UV detection at 280 nm, and fractions were analyzed by SDS-PAGE to assess purity.

Used instrumentation

• AZURA® P6.1L HPG metal-free pump
• AZURA® ASM 2.1L assistant module with multi-position valves
• AZURA® MWD2.1L multi-wavelength detector (280 nm)
• Tricorn™ 10/200 column packed with Chelating Sepharose™ Fast Flow (15 mL)
• Semi-preparative biocompatible flow cell (3 mm path, 2 µL volume)

Results and discussion

IMAC separation yielded a distinct elution peak corresponding to His-tagged SOx, while non-bound proteins appeared in the flow-through. SDS-PAGE analysis confirmed a prominent band at ~15 kDa in the eluted fraction, consistent with the expected molecular weight. Contaminant bands were minimal, indicating high purity. A standard preparation achieved approximately 1.13 g/L of purified enzyme.

Benefits and practical applications of the method

The established protocol enables efficient isolation of high-purity SOx, facilitating downstream biochemical characterization and stability testing. In food processing, SOx can serve as a biological alternative to chemical cross-linkers, enhancing texture and shelf-life in protein-based products.

Future trends and opportunities

Scaling up production and integrating continuous chromatography systems may increase throughput and cost efficiency. Protein engineering could further improve SOx stability and activity under industrial conditions. Expanding applications beyond food stabilization to biotechnology and pharmaceutical protein folding represents additional avenues for development.

Conclusion

The affinity chromatography method using an AZURA® Bio system provides a reproducible and high-yield purification of recombinant sulfhydryl oxidase. This advancement supports comprehensive enzyme characterization and paves the way for its adoption as a natural stabilizer in the food industry.

References

• Trivedi MV, Laurence JS, Siahaan TJ. The role of thiols and disulfides on protein stability. Curr Protein Pept Sci. 2009;10(6):614–625.