Purification, Biochemical Characterization, and Facile Immobilization of Laccase from Sphingobacterium ksn - 11 and its
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Purification, Biochemical Characterization, and Facile Immobilization of Laccase from Sphingobacterium ksn-11 and its Application in Transformation of Diclofenac Kulkarni S. Neelkant 1 & Kumar Shankar 1 & Senigala K. Jayalakshmi 2 & Kuruba Sreeramulu 1 Received: 13 March 2020 / Accepted: 23 June 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
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 azinobis (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. Keywords Laccase . Purification . Immobilization . Diclofenac transformation . LC-MS
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12010-02003371-1) contains supplementary material, which is available to authorized users.
* Kuruba Sreeramulu [email protected]; [email protected] Extended author information available on the last page of the article
Applied Biochemistry and Biotechnology
Introduction Laccase (benzenediol: oxygen oxidoreductases; EC 1.10.3.2) is an important class of enzymes, which has become remarkable scientific interest due to its greater catalytic capability to various phenolic and non-phenolic compounds with simultaneous reduction of oxygen to water molecule. The catalytic domain consists of three copper redox sites (T1, T2, and T3) [1, 2]. T1 site is involved in the oxidation of substrates; meanwhile, T2 and T3 are involved in a trinuclear cluster formation for electron movement by molecular reduction. This distinct property makes this enzyme versatile and employed in various industrial and environmental applications such as cosmetics, bioremediation, biofuel, and biosensor technology [3, 4]. The persistent studies on many fungal laccases have been vastly characterized and utilized for effluent azo dye detoxification [5], textile, and in production of industrially important compounds [6]. Bacterial laccases, on the other hand, gained focus, and genomic analysis revealed that enzymes are wide spread in
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