Vegetos
(An International Journal of Plant Research & Biotechnology)
Cloning of laccase gene from Coriolopsis caperata into heterologous E.coli host
Research Articles | Published: 02 December, 2022
Online ISSN : 2229-4473.
Website:www.vegetosindia.org
Pub Email: contact@vegetosindia.org
First Page: 195
Last Page: 200
Views: 747
Keywords:
Laccase gene,
E.coli
,
Coriolopsis caperata
, Directional cloning, Industrial enzymes
Abstract
The cloning of the laccase gene encoding industrially relevant laccase enzyme was carried out from Coriolopsis caperata into heterologous E.coli. Laccase enzymes play a role in the oxidation reactions of one electron of a wide range of phenols, aromatic amines and heterocyclic compounds such as lignin. They are multi-copper oxidoreductases that catalyze the one-electron (e-) oxidation coupled with the transfer of four electrons to the catalytic copper atoms, which leads to the reduction of two water molecules from reducing oxygen. In the present study, C. caperata was utilized for cloning the laccase gene and the protocol for cloning is outlined in the present study. The full-length gene (~ 1622bp) was amplified from Coriolopsis caperata by extracting RNA and synthesizing cDNA and PCR using gene-specific primers. By including the restriction site sequence of NcoI and XhoI enzymes into the synthetic primers, the directed cloning of the laccase gene fragment in the pET28b vector was performed.The construct was transformed into the E.coli BL-21 DE3 strain for expression. The transformed E.coli strain was screened on the kanamycin marker plate and the recombinant colonies were selected for plasmid isolation. Laccase gene cloning was confirmed by DNA sequencing of extracted plasmid. The cells were further processed for inclusion bodies isolation for enzyme recovery. The protein obtained was of size ~ 57kDa and confirmed on SDS PAGE which was same as expected size. The enzyme activity was found to be 73.1U/mL using ABTS as substrate. The laccase gene was successfully cloned from the fungus C. caperata and because laccase enzyme has widespread applications in the pharmaceutical, food, and environmental industries the E.coli recombinant host expressing laccase gene could serve as a warehouse for higher laccase enzyme production in these industries.
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Acknowledgements
I would like to thank geneOmbio Technologies Pvt Ltd, Pune for the providing the laboratory facility required for the lab work.
Author Information
Rajiv Gandhi Institute of Information Technology and Biotechnology, Bharati Vidyapeeth Deemed to be University, Pune, Maharashtra, India
manasee.dw@gmail.com
Rajiv Gandhi Institute of Information Technology and Biotechnology, Bharati Vidyapeeth Deemed to be University, Pune, Maharashtra, India