A Novel Two-Domain Laccase with Middle Redox Potential: Physicochemical and Structural Properties

Area of Science:

• Biochemistry and Molecular Biology
• Enzymology
• Microbial Biotechnology

Background:

• Laccases are multi-copper oxidases with diverse industrial applications.
• Two-domain laccases are a distinct subclass, but their properties are less explored.
• Streptomyces species are known sources of novel enzymes.

Purpose of the Study:

• To identify and characterize a novel two-domain laccase from Streptomyces carpinensis.
• To investigate the enzyme's biochemical properties, including activity, stability, and redox potential.
• To elucidate the structural basis for its unique characteristics.

Main Methods:

• Gene identification and heterologous expression in Escherichia coli.
• Enzyme purification using affinity chromatography.
• Biochemical assays for activity, stability, pH optima, and substrate specificity.
• Determination of kinetic parameters (Km, kcat) and redox potential.
• X-ray crystallography for structural analysis.

Main Results:

• A novel two-domain laccase, ScaSL, was successfully expressed and purified.
• ScaSL exhibited unique activity in multimeric forms, alongside homotrimer activity.
• The enzyme demonstrated high thermal stability (optimal activity at 80°C) and broad pH stability (pH 9-11).
• ScaSL catalyzed the polymerization of humic acids and lignin.
• The determined redox potential (0.472 ± 0.007 V) is classified as middle, a first for two-domain laccases.
• Crystal structure revealed potential structural basis for middle redox potential related to amino acid residue 230.

Conclusions:

• ScaSL is the first characterized two-domain laccase with a middle redox potential.
• Its unique properties, including thermal stability and multimeric activity, make it a promising candidate for biotechnological applications.
• Structural insights provide a foundation for understanding and engineering laccase function.