Simultaneous purification and immobilization of soybean hull peroxidase with a dye attached to chitosan mini-spheres

Abstract

Soybean hull peroxidase (EC 1.11.1.7, SBP) was simultaneously purified and immobilized by dye affinity chromatography with Reactive Blue 4 attached to chitosan mini-spheres. Under optimized conditions, 96% of SBP was adsorbed to the matrix. Under the most stringent condition, only 49% was desorbed, whereas 2 M NaCl failed to desorb a significant amount of SBP. This behaviour allowed proposing the dye matrix as a support to immobilize SBP from a crude extract. The pH of maximum activity shifted from 7 to 3–5. SBP gained thermostability after immobilization: after 5 h at 85 °C, the remaining activity was 54%, whereas that of the free enzyme was 31%. The optimum temperature for the immobilized SBP was 75 °C, whereas that of the free enzyme was 55 °C. After two months at 4 °C, the activity loss of the immobilized SBP was only 3%. Immobilized SBP removed 80% of 2-bromophenol from wastewater in 180 min and, after five cycles of use, the activity loss was only 12.8%.

Keywords:

• Soybean peroxidase
• purification
• immobilization
• affinity chromatography
• triazine dyes
• bioremediation

Disclosure statement

This work was supported by a grant from the Agencia Nacional de Promoción Científica y Técnica de la República Argentina [PICT 2012 – 1881].

GJl, FJW, MVM and OC are career researchers of the CONICET. LFB is a fellow of the CONICET.

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by a grant from the Agencia Nacional de Promoción Científica y Técnica de la República Argentina [PICT 2012 – 1881].