AN EXHAUSTIVE EXAMINATION AND ASSESSMENT OF THE REPLICATION AND RECOMBINANT PRODUCTION OF A HEAT-RESISTANT LACCASE ENZYME IN ESCHERICHIA COLI

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Muhammad Raza
Momal Maqsood
Sana Hussain
Ali Zia
Farhan Afzal
Kainat Khalid
Sawaira Ahmad

Keywords

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Abstract

Laccase is an enzyme known for its ability to expedite chemical reactions. It belongs to a specific class of enzymes that naturally generate reactive radicals, which have applications in various biological processes and find use in commercial applications across chemical and biotechnological industries. Laccases are not limited to bacteria; they are also found in different species of fungi and plants. Their industrial applications are diverse, including the detoxification of industrial effluents, utilization in cosmetic and pulp and paper industries, and medical applications such as diagnostic tools in laboratories and in the development of anti-cancer drugs. They are also used in water purification processes. The objective of this current research is to ascertain the production of recombinant laccase in Escherichia coli and optimize the laccase parameters to achieve the fastest possible expression of the enzyme. Initially, recombinant laccase will be produced on a laboratory scale, and its expression will be analyzed. The laccase gene will be obtained from the thermophilic bacterium Clostridium thermocellum. Escherichia coli and BL21 will be employed for cloning, vector propagation, and observing the expression of the recombinant laccase enzyme in Escherichia coli. Primers will be designed by retrieving the gene sequence from the NCBI database and designing them using the Primer-3 input software (v. 0.4.0) based on the coding region. Restriction enzymes will be chosen using the NEB cutter (V2.0) tool. DNA isolation will be carried out using the DNA chloroform method, and its quality will be verified through 1% agarose gel electrophoresis. DNA isolation will be followed by PCR amplification and confirmation on 1% agarose gel. The purified laccase gene fragments, F1 and F2, will be cloned into the expression vectors pET22b(+) and pET28b(+) through single digestion with NdeI and double digestion of the plasmid with NcoI and XhoI. E. coli BL21 codonPlus cells will be rendered competent using the CaCl2 method, and the cloned DNA will be introduced into the competent cells via the heat shock method. Shake flask fermentation will be conducted by inducing the cells with lactose and IPTG as inducers in LB media, and their expression will be assessed using 12% SDS-PAGE.During this study, the effects of various influential parameters on the kinetics of the laccase enzyme will also be investigated. Optimization and the study of reaction kinetics will involve using different substrates, such as ABTS, Syringaldazine, or HOBT.

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