Production and Partial Characterization of Collagenase from Rhizopus microsporus UCP 1296: Cytotoxic Potential and Controlled Release Strategy

Abstract

Fungal species, especially those from underexplored ecological niches, offer a promising avenue for novel enzyme discovery due to their metabolic versatility and ability to secrete enzymes with favorable industrial properties. However, the biotechnological potential of fungi from extreme or unique biomes, such as the semi-arid Caatinga in Brazil, remains largely untapped. Moreover, although some fungal collagenases have been studied for therapeutic use, there is a lack of comprehensive research integrating the optimization of production steps and the development of efficient delivery systems. This study selected a new strain of Rhizopus microsporus (UCP 1296), isolated from soil in the Caatinga, a Brazilian biome, for collagenase production. A 2⁴-Full Factorial Design and a 2²-Central Composite Design, combined with the Response Surface Methodology (RSM), were used to optimize collagenase production in submerged liquid culture. Subsequently, the physicochemical characterization of the enzyme and its incorporation into galactomannan gel were performed. The combined application of statistical designs and RSM allowed for a 63% increase in collagenase production (872 ± 43 U/mg). The enzyme showed maximum activity at pH 8.0 and 40 °C, maintaining stability across a wide range of pH and temperature. It was strongly inhibited by phenylmethylsulphonyl fluoride (PMSF) and was capable of hydrolyzing type I collagen and azocoll. The experiments of collagenase incorporation in galactomannan gel demonstrated that no less than 72% of the enzyme was retained in the matrix, maintaining high activity after 24 h (305.11 U/mL). Furthermore, collagenase was released from the gel following a pseudo-Fickian behavior and did not exhibit cytotoxic effects on L929 fibroblasts, confirming its biocompatibility and suitability as a controlled release system. These results represent an advancement in the sustainable production of fungal collagenase and its incorporation into a galactomannan-based gel system, with potential for large-scale application in the pharmaceutical and cosmetic industries.