Experimental immobilization of Clostridium histolyticum collagenases on green-synthesized CuO nanoparticles supported...

Enzymes are widely used in various industries due to their eco-friendly nature, high selectivity, and ability to operate under mild conditions. However, large-scale applications are often hindered by challenges such as limited stability, high costs, and difficulties in recyclability. Enzyme immobilization presents a promising strategy to overcome these limitations. Microbial collagenases, particularly those from Clostridium histolyticum, play a crucial role in industrial and biomedical applications. In silico analyses effectively predict enzyme immobilization outcomes, minimizing experimental time and cost. This study employed enzyme surface analysis, a bioinformatics approach, to determine the surface charge type of the support for the physical immobilization of C. histolyticum collagenases. The analysis identified the most suitable regions on the enzyme surfaces for attachment-positively charged and distant from critical sites-ensuring minimal disruption to activity. Copper oxide (CuO) nanoparticles (NPs) were synthesized using walnut green husk extract and subsequently characterized. The green-synthesized CuO NPs were monodisperse and spherical (∼40 nm) with a negatively charged surface, confirming their suitability as supports for enzyme immobilization. Employing the CuO NPs as supports resulted in an immobilization yield of 42.15 % and an activity yield of 146.2 %. Further optimization of immobilization conditions could improve IY. The high AY suggests that immobilization did not hinder enzyme function but may have enhanced enzyme-substrate affinity and increased local substrate concentration, thereby boosting enzyme efficiency.