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Molecular and Computational Approaches to Characterize Thermostable Laccase Gene from Two Xerophytic Plant Species

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Abstract

Laccases are blue multicopper oxidases that carry out single electron transfers in the oxidation of phenols to quinones. In plants, they confer structural stability to the cell wall. Thermostable laccases were identified in xerophytes Cereus pterogonus and Opuntia vulgaris that could be used in biotechnology and industrial processes. Polyclonal anti-laccase antibodies were generated against purified laccase enzyme isoforms capable of 98–99 % inhibition of the catalytic activity. Antibodies raised against lower molecular weight isoforms inhibited 70 % of the catalytic activity of higher molecular forms. Only 20 % inhibition was noted when assayed in reverse. A partial gene sequence of thermostable xerophytic laccase comprising 712 and 880 bp was identified employing cDNA as template. The nucleotide sequence was submitted to GenBank. The gene sequence was in silico translated into protein sequence and a 3-D structure was predicted using I-Tasser and Genesilico online servers that justified the experimental observations. Anti-laccase antibodies and nucleotide gene sequence of this thermostable plant laccase can be utilized for predicting laccase antigenic sequences and for cloning and expression of the thermostable eukaryotic laccase.

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Acknowledgments

The authors wish to thank Mr. B. Ramachandra Mouli, Research Scholar, Molecular Medicine, Hannover Medical School, Hannover, Germany for the support rendered in generating polyclonal antibodies and Mr. K. Suresh, Research Scholar, AJOU University, Korea for timely suggestions in designing primers and in carrying out computational studies. Gali Nirmal Kumar wishes to thank University Grants Commission, New Delhi, India for the award of UGC Meritorious Research Fellowship in Science.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Puducherry, 605014, India

    Gali Nirmal Kumar & Kotteazeth Srikumar

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  1. Gali Nirmal Kumar
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  2. Kotteazeth Srikumar
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Correspondence to Kotteazeth Srikumar.

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Ethical Considerations

The experiments with animal models were carried out carefully as per “Committee for the Purpose of Control and Supervision of Experiments on Animals” (CPCSEA 2003), New Delhi, India, guidelines and approved by Institutional Ethical Committee of Pondicherry University, Puducherry, India. The experimentation details were duly registered in animal house facility maintained at Pondicherry University, Puducherry, India. Animals were used to raise polyclonal antibodies, followed by careful drawing of blood, and at the end, animals were rehabilitated as per the guidelines.

Accession numbers in the GenBank database are as follows: JN793991, JN793990, 84618776, 30679958, 1621460, 3805959, and 13661206.

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Kumar, G.N., Srikumar, K. Molecular and Computational Approaches to Characterize Thermostable Laccase Gene from Two Xerophytic Plant Species. Appl Biochem Biotechnol 172, 1445–1459 (2014). https://doi.org/10.1007/s12010-013-0611-6

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  • DOI: https://doi.org/10.1007/s12010-013-0611-6

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