Purification, Biochemical Characterization, and Facile Immobilization of Laccase from Sphingobacterium ksn-11 and its Application in Transformation of Diclofenac


An extracellular laccase enzyme secreted from Sphingobacterium ksn-11 was purified to electrophoretic homogeneity, showing a molecular weight of 90 kDa. The purified enzyme was monomeric in nature confirmed by sodium dodecyl gel electrophoresis. The optimum temperature and pH were found to be 40 °C and 4.5 respectively. The enzyme showed highest substrate specificity for 2,2 azino-bis (ethylthiozoline-6-sulfonate) (ABTS), followed by syringaldazine. The Km value for ABTS was 2.12 mM with a Vmax value of 33.33 U/mg which was higher when compared with syringaldazine and guaiacol substrates. Sodium azide and EDTA inhibited the activity by 30%, whereas presence of Ca2+ and iron increased activity by 50%. The purified enzyme was immobilized in sodium alginate-silicon dioxide-polyvinyl alcohol beads and evaluated for diclofenac transformation studies. LC-MS analysis confirmed that immobilized laccase transformed diclofenac to 4-OH diclofenac after 4 h of incubation. 45 % of diclofenac was able to transform even at 3rd cycle of immobilized laccase use. Therefore, immobilized laccase can be used to transform or degrade several recalcitrant compounds from industrial effluents.

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The authors are grateful and acknowledge SAIF IIT Bombay for LC-MS analysis.


This study is funded by UGC-SAP programme DRS-II for Dept of Biochemistry Gulbarga University.

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Correspondence to Kuruba Sreeramulu.

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The original version of this article was revised: The original version of this article unfortunately contained a mistake in the equation under “Immobilized Laccase Activity and its Storage Stability” section.

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Neelkant, K.S., Shankar, K., Jayalakshmi, S.K. et al. Purification, Biochemical Characterization, and Facile Immobilization of Laccase from Sphingobacterium ksn-11 and its Application in Transformation of Diclofenac. Appl Biochem Biotechnol (2020). https://doi.org/10.1007/s12010-020-03371-1

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  • Laccase
  • Purification
  • Immobilization
  • Diclofenac transformation
  • LC-MS