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Optimisation of the Production and Bleaching Process for a New Laccase from Madurella mycetomatis, Expressed in Pichia pastoris: from Secretion to Yielding Prominent

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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.

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  • 12 November 2020

    In the paper by “Tülek, A., Karataş, E., Çakar, M. M., Aydın, D., Yılmazcan, Ö, Binay, B. (2020). Optimisation of the Production and Bleaching Process for a New Laccase from Madurella mycetomatis, Expressed in Pichia pastoris: from Secretion to Yielding Prominent Performance. <Emphasis Type="Italic">Molecular Biotechnology</Emphasis>, <ExternalRef><RefSource>https://doi.org/10.1007/s12033-020-00281-9</RefSource><RefTarget Address="10.1007/s12033-020-00281-9" TargetType="DOI"/></ExternalRef>”

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Acknowledgements

The authors gratefully acknowledge the help and support of ETDAM (Enzyme Testing and Consultation Center, Gebze Technical University Turkey).

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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Gebze Technical University, 41400, Gebze, Kocaeli, Turkey

    Ahmet Tülek, Ersin Karataş & Mehmet Mervan Çakar

  2. Ak-Kim Kimya San. Ve Tic. A.S., 77600, Çiftlikköy, Yalova, Turkey

    Derya Aydın & Özlem Yılmazcan

  3. Department of Bioengineering, Gebze Technical University, 41400, Gebze, Kocaeli, Turkey

    Barış Binay

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  1. Ahmet Tülek
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  2. Ersin Karataş
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  3. Mehmet Mervan Çakar
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  4. Derya Aydın
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  5. Özlem Yılmazcan
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  6. Barış Binay
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Contributions

BB designed the study. AT, EK, MMÇ, DA, ÖY performed the study. AT, EK, MMÇ, DA, ÖY and BB analysed the data. AT and BB wrote the paper. All authors read and approved the manuscript.

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Correspondence to Barış Binay.

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Tülek, A., Karataş, E., Çakar, M. M., Aydın, D., Yılmazcan, Ö., & Binay, B. (2020). Optimisation of the production and bleaching process for a New Laccase from Madurella mycetomatis, expressed in Pichia pastoris: From Secretion to Yielding Prominent Performance. Molecular Biotechnology. https://doi.org/10.1007/s12033-020-00281-9.

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Fig. S1 Multiple amino acid sequence alignments of laccases. Percentages indicate the sequence identity between MmLac and isozyme laccases (Mm: Madurella mycetomatis; Ta: Thielavia arenaria; Ma: Melanocarpus albomyces; Mt: Myceliophthora thermophila; Ba: Botrytis aclada and An: Aspergillus niger). The orange, magenta, blue, red and green rectangles indicate deleted MmLac native signal sequences, N-glycosylation sites, Cu2+ binding sites, catalytic residues of the MmLac active site pocked and deleted MmLac native propeptide sequence, respectively. Fig. S2 Amino acid sequence and functional regions of fusion MmLac. The native N-terminal signal peptide and native pro-peptide are shown in orange, the MF-α signal in grey, N-terminal extension sequence (ETEAEF) in cyan, polyhistidine tag (6xHis-tag) in yellow, MmLac in claret red and C-terminal pro-peptide in green. Fig. S3 Screening and production of extracellular MmLac in MM agar plates: (a) Control plate; (b) Pichia pastoris X33 containing mmlac1 construct; (c) Pichia pastoris X33 containing mmlac2 construct. Fig. S4 Michaelis-Menten plot of MmLac in presence of (a) ABTS; (b) DMP; (c) SGZ. Fig. S5 MALDI-TOF mass spectrum of the purified MmLac laccase: a) Purified MmLac with glycosylation site (67.4 kDa); b) Deglycosylated laccase (62.0 kDa).

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Tülek, A., Karataş, E., Çakar, M.M. et al. Optimisation of the Production and Bleaching Process for a New Laccase from Madurella mycetomatis, Expressed in Pichia pastoris: from Secretion to Yielding Prominent. Mol Biotechnol 63, 24–39 (2021). https://doi.org/10.1007/s12033-020-00281-9

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