Recombinant laccase: I. Enzyme cloning and characterization.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Laccases are copper-containing oxidases with diverse industrial and medical applications.
• Rigidoporus lignosus is a white-rot basidiomycete known to produce laccase enzymes.
• Efficient expression and purification are crucial for characterizing and utilizing recombinant laccases.

Purpose of the Study:

• To optimize the expression and purification of a recombinant Laccase from Rigidoporus lignosus.
• To characterize the functional properties of the purified recombinant Laccase.
• To assess the potential of this Laccase for enzymatic biosensor development.

Main Methods:

• Laccase coding sequence amplified via PCR from cDNA.
• Cloning into a bacterial expression system (pET-28b plasmid) with T7/Lac promoter.
• Expression in supercompetent Escherichia coli cells and induction with IPTG.
• Purification using fast protein liquid chromatography (FPLC).
• Functional characterization using circular dichroism (CD) spectra, cyclic voltammetry (CV), and biochemical assays.

Main Results:

• Successfully optimized expression and purification protocols for recombinant Laccase.
• Demonstrated functional activity of the purified enzyme through electrochemical measurements.
• Showcased compatibility with Syringaldazine (SGZ) for potential biosensor applications.

Conclusions:

• The optimized expression and purification yield a functional recombinant Laccase from Rigidoporus lignosus.
• The characterized Laccase is suitable for further development into enzymatic biosensors.
• This work lays the foundation for novel medical diagnostic tools using laccase-based biosensors.