Optimisation of the Production and Bleaching Process for a New Laccase from Madurella mycetomatis , Expressed in Pichia
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ORIGINAL PAPER
Optimisation of the Production and Bleaching Process for a New Laccase from Madurella mycetomatis, Expressed in Pichia pastoris: from Secretion to Yielding Prominent Ahmet Tülek1 · Ersin Karataş1 · Mehmet Mervan Çakar1 · Derya Aydın2 · Özlem Yılmazcan2 · Barış Binay3 Accepted: 7 October 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Laccases are polyphenol oxidoreductases used in a number of industrial applications. Due to the increasing demand for these “green catalysis” enzymes, the identification and biochemical characterisation of their novel properties is essential. In our study, cloned Madurella mycetomatis laccase (mmlac) genes were heterologously expressed in the methylotrophic yeast host Pichia pastoris. The high yield of the active recombinant protein in P. pastoris demonstrates the efficiency of a reliably constructed plasmid to express the laccase gene. The optimal biochemical conditions for the successfully expressed MmLac enzyme were identified. Detailed structural properties of the recombinant laccase were determined, and its utility in decolourisation and textile bleaching applications was examined. MmLac demonstrates good activity in an acidic pH range (4.0–6.0); is stable in the presence of cationic metals, organic solvents and under high temperatures (50–60 °C); and is stable for long-term storage at − 20 °C and − 80 °C for up to eight weeks. The structural analysis revealed that the catalytic residues are partially similar to other laccases. MmLac resulted in an increase in whiteness, whilst demonstrating high efficiency and stability and requiring the input of fewer chemicals. The performance of this enzyme makes it worthy of investigation for use in textile biotechnology applications, as well as within environmental and food technologies. Keywords Madurella mycetomatis · Laccase · Stability · Structural modelling · Pichia pastoris · Textile bleaching Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12033-020-00281-9) contains supplementary material, which is available to authorized users. * Barış Binay [email protected] Ahmet Tülek [email protected] Ersin Karataş [email protected] Mehmet Mervan Çakar [email protected] Derya Aydın [email protected] Özlem Yılmazcan [email protected] 1
Department of Molecular Biology and Genetics, Gebze Technical University, 41400 Gebze, Kocaeli, Turkey
2
Ak-Kim Kimya San. Ve Tic. A.S., 77600 Çiftlikköy, Yalova, Turkey
3
Department of Bioengineering, Gebze Technical University, 41400 Gebze, Kocaeli, Turkey
Introduction Laccases (EC 1.10.3.2, benzenediol: O 2 oxidoreductases) belong to the multi-copper superfamily of enzymes, often used as biocatalysts in industrial biotechnology. These enzymes form water by transferring the electrons released from the oxidation of phenols, non-phenols, amines, anilines and other compounds to molecular oxygen [1]. Structurally, laccases as a member of the cuppredoxin superfamily, are known t
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