# Altering the Properties of Laccases from Ensifer meliloti (Sinorhizobium meliloti) and Cerrena unicolor by Chemical Modifications of Proteins

## TL;DR

This study explores how chemical modifications affect the structure and function of laccases from two different sources, aiming to improve their stability and activity for biotechnology applications.

## Contribution

The novel contribution is the comparative characterization and chemical modification of laccases from Ensifer meliloti and Cerrena unicolor to enhance their biotechnological potential.

## Key findings

- Chemical modifications significantly increased the activity of bacterial and fungal laccases by up to 19%.
- Modifications with GA-CDI and CA were most effective in improving thermal stability of the enzymes.
- Certain modified enzymes retained full activity in the presence of high concentrations of NaI and NaCl.

## Abstract

Due to their catalytic performance, laccases constitute one of the most promising groups of enzymes for potential applications in modern biotechnology. In this study, we aimed to chemically modify Ensifer meliloti (Sinorhizobium meliloti) and Cerrena unicolor laccase and comparatively characterize the structures of both enzymes. The most characteristic feature was the spatial localization of lysine residues, predominantly positioned distal to the active site region for both compared enzymes. The solvent-accessible surface area (SASA) analysis showed that bacterial laccase was characterized by a larger hydrophobic SASA than the fungal enzyme. The pKa prediction identified only one Lys in the E. meliloti laccase structure susceptible to modification. Modifications were achieved by using mono- and bifunctional crosslinking agents, and glycosylations were also performed. The degree of protein modification ranged from 0% for glucose- and galactose-modified E. meliloti laccase and citraconic anhydride-modified (CA) C. unicolor laccase to 62.94% for the palmitic acid N-hydroxysuccinimide ester-modified E. meliloti enzyme. The stability of covalently modified laccases over a wide pH and temperature ranges and in the presence of inhibitors was investigated. Protein modifications with polymeric sucrose (PS) and ethylene glycol bis-(succinimidyl succinate) (EGNHS) significantly increased the activity of the bacterial and fungal laccases by 15 and 19%, respectively. Although pH optima remained relatively unchanged by modifications, certain variants, especially CA-modified bacterial protein and EGNHS-modified C. unicolor enzyme, exhibited improved stability at near-neutral pH (6–7). Modification of the bacterial enzyme with glutaraldehyde-carbodiimide (GA-CDI-ver) and of the fungal enzyme with CA was the most effective in improving its thermal stability. Chemical modifications using GA, CDI, GA-CDI, and PS allowed E. meliloti L 3.8 laccase to retain full activity in the presence of 5 mM NaI, whereas CA-, PS-, and EGNHS-modified C. unicolor variants retained their activity even at elevated NaCl concentrations. The results clearly demonstrate that the outcome of chemical modifications is closely linked to enzyme-specific structural features and that selecting an appropriate modification strategy is critical to achieving the desired effect.