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

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 <i>Ensifer meliloti</i> (<i>Sinorhizobium meliloti</i>) and <i>Cerrena unicolor</i> 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 pK_a prediction identified only one Lys in the <i>E. meliloti</i> 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 <i>E. meliloti</i> laccase and citraconic anhydride-modified (CA) <i>C. unicolor</i> laccase to 62.94% for the palmitic acid N-hydroxysuccinimide ester-modified <i>E. meliloti</i> 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 <i>C. unicolor</i> 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 <i>E. meliloti</i> L 3.8 laccase to retain full activity in the presence of 5 mM NaI, whereas CA-, PS-, and EGNHS-modified <i>C. unicolor</i> 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.