Expression of thermophilic two-domain laccase from Catenuloplanes japonicus in Escherichia coli and its activity against triarylmethane and azo dyes
| Autor: | S. V. Alferov, O. N. Ponamoreva, Alexey Arkadyevich Leontievsky, L.I. Trubitsina, Anna Petrovna Larionova, Ivan Vasilyevich Trubitsin, Azat Vadimovich Abdullatypov |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
0301 basic medicine Biochemistry Microbiology 01 natural sciences Syringaldehyde General Biochemistry Genetics and Molecular Biology Ferulic acid 03 medical and health sciences chemistry.chemical_compound Affinity chromatography Caffeic acid Molecular Biology Laccase Catenuloplanes japonicus ABTS Triphenylmethane Hydroquinone General Neuroscience General Medicine 030104 developmental biology chemistry Dye decolorization Enzymology Medicine Heterologous expression General Agricultural and Biological Sciences Biotechnology 010606 plant biology & botany Nuclear chemistry |
| Zdroj: | PeerJ, Vol 9, p e11646 (2021) PeerJ |
| ISSN: | 2167-8359 |
| Popis: | Background Two-domain laccases are copper-containing oxidases found in bacteria in the beginning of 2000ths. Two-domain laccases are known for their thermal stability, wide substrate specificity and, the most important of all, their resistance to so-called «strong inhibitors» of classical fungal laccases (azides, fluorides). Low redox potential was found to be specific for all the two-domain laccases, due to which these enzymes lost the researchers’ interest as potentially applicable for various biotechnological purposes, such as bioremediation. Searching, obtaining and studying the properties of novel two-domain laccases will help to obtain an enzyme with high redox-potential allowing its practical application. Methods A gene encoding two-domain laccase was identified in Catenuloplanes japonicus genome, cloned and expressed in an Echerichia coli strain. The protein was purified to homogeneity by immobilized metal ion affinity chromatography. Its molecular properties were studied using electrophoresis in native and denaturing conditions. Physico-chemical properties, kinetic characteristics, substrate specificity and decolorization ability of laccase towards triphenylmethane dyes were measured spectrophotometrically. Results A novel two-domain recombinant laccase CjSL appeared to be a multimer with a subunit molecular mass of 37 kDa. It oxidized a wide range of phenolic substrates (ferulic acid, caffeic acid, hydroquinone, catechol, etc.) at alkaline pH, while oxidizing of non phenolic substrates (K4[Fe(CN)6], ABTS) was optimal at acidic pH. The UV-visible absorption spectrum of the purified enzyme was specific for all two-domain laccases with peak of absorption at 600 nm and shoulder at 340 nm. The pH optima of CjSL for oxidation of ABTS and 2, 6-DMP substrates were 3.6 and 9.2 respectively. The temperature optimum was 70 °C. The enzyme was most stable in neutral-alkaline conditions. CjSL retained 53% activity after pre-incubation at 90 °C for 60 min. The enzyme retained 26% activity even after 60 min of boiling. The effects of NaF, NaN3, NaCl, EDTA and 1,10-phenanthroline on enzymatic activity were investigated. Only 1,10-phenanthroline reduced laccase activity under both acidic and alkaline conditions. Laccase was able to decolorize triphenylmethane dyes and azo-dyes. ABTS and syringaldehyde were effective mediators for decolorization. The efficacy of dye decolorization depended on pH of the reaction medium. |
| Databáze: | OpenAIRE |
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