Collagenases are enzymes with broad biotechnological applications in medicine. This study describes the production and characterization of a collagenase from Aspergillus serratalhadensis URM 7866, isolated from the Caatinga biome. Solid-state fermentations were conducted using wheat bran under varying conditions of pH (6, 7, 8), moisture content (50%, 60%, 70%), and substrate concentration (2.5 g, 5 g, 10 g). The optimal condition—10 g of wheat bran at pH 8 and 70% moisture—yielded the highest collagenolytic activity (177.96 U/mL) and a specific activity of 50.55 U/mg. The enzyme was purified via multiple chromatography, with pre-purification and final purification factors of 18.09 and 20.21, respectively, reaching a specific activity of 1021.86 U/mg. The enzyme showed optimal activity at 50 °C and pH 8, with stability from 20 to 40 °C and pH 7–9. PMSF caused >80% inhibition; EDTA caused ~34% inhibition. Activity increased with Na⁺ and Ca²⁺ and was inhibited by Zn²⁺. The enzyme retained full activity in anionic and non-ionic surfactants (1–10%). FTIR confirmed characteristic amide bands, and kinetic analysis revealed a Km of 1.72 mg/mL and Vmax of 6.89 mg/mL/min. These findings support its potential for alkaline and surfactant-rich industrial processes.

Whey, a large-scale dairy industry by-product, can be converted into whey protein concentrate (WPC), providing a cost-effective nutrient-rich substrate for microbial fermentation. We investigated protease production by Aspergillus oryzae using WPC as the sole substrate in submerged fermentation. Following fermentation, the protease was purified sequentially from the crude extract by salting-out, which yielded a substantial purification factor (~39), and subsequent ion-exchange chromatography. The non-adsorbed chromatographic fraction showed the highest protease activity (92.6 U/mL) and revealed one main protein band ~45 kDa via SDS-PAGE. Enzyme characterization demonstrated activity across neutral-to-alkaline conditions, optimal at pH 9.0 and 37 °C, with stability maintained between 30 °C and 37 °C. The enzyme was classified as a serine protease based on strong inhibition by PMSF and SDS; its activity was also inhibited by Zn²⁺, Mg²⁺, and K⁺, but enhanced by Ca²⁺. This work validates WPC as an efficient substrate for protease production by A. oryzae and presents a promising strategy for valorizing industrial by-products through sustainable biotechnology.

O gênero Trichoderma é amplamente reconhecido por seu potencial biotecnológico, des-tacando-se na produção de enzimas e na promoção do controle biológico, o que o torna um recurso valioso para processos industriais sustentáveis. Este estudo investigou a pro-dução de proteases colagenolíticas pelo Trichoderma koningiopsis (UFPIT03) cultivado em fermentação em estado sólido (FES) utilizando farelo de frutos de Pritchardia paci-fica como substrato. O principal objetivo foi explorar o potencial desses resíduos agroin-dustriais para a obtenção de enzimas com aplicação industrial. As proteases foram extra-ídas e submetidas a um esquema de purificação que incluiu precipitação com acetona, fracionamento com sulfato de amônio e cromatografia de troca iônica. Os resultados obtidos apresentaram uma eficiência na purificação de uma enzima com atividade prote-olítica exibindo atividade ótima em pH 8,0 e estabilidade térmica, mantendo mais de 70% da atividade após 6 horas de incubação em temperaturas entre 20°C e 50°C. Adici-onalmente, a caracterização físico-química indicou que a enzima na presença de íons AlCl3 e ZnSO4 teve sua atividade reduzida em mais de 50% e estabilidade na presença de surfactantes. A cromatografia em Gel filtração em Superdex-G75 revelou a massa molecular da enzimas em torno de 65kDa. Os resultados demonstram a viabilidade do T. koningiopsis como recurso biotecnológico para a obtenção de enzimas de alto valor agregado, promovendo processos sustentáveis que aproveitam resíduos agroindustriais.

Anais do Congresso Internacional em Saúde e Desenvolvimento Socioambiental (978-65-272-0801-3) - Potencial Do Fungo Aspergillus Ochraceus Urm604 Para Produção De Enzimas De Interesse Farmacêutico

The contamination of water bodies by industrial dyes is a critical environmental challenge due to the toxicity and persistence of these compounds in aquatic ecosystems. This study evaluated the efficiency of Trichoderma koningiopsis immobilized on Luffa cylindrica matrices for the decolorization of the azo dye Direct Black 22 (DB22), proposing a biotechnological approach for wastewater treatment. The fungus was cultivated and immobilized on matrices characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Experiments under different temperature, pH, and initial dye concentration conditions demonstrated that the immobilized system achieved up to 96% decolorization within 24 h under optimized conditions of 50 °C and pH 4, significantly outperforming the free fungus. The Luffa cylindrica matrix provided mechanical stability and a larger contact area for DB22 decolorization. Thus, the immobilized Trichoderma koningiopsis system on Luffa cylindrica stands out as a sustainable, cost-effective, and efficient alternative for dye removal from textile effluents, contributing to safer and more effective environmental practices.

Proteases are among the most commercially important industrial enzymes, yet their large-scale production is often limited by complex and costly downstream processing. In this study, an integrated bioprocess was developed for the production, in situ recovery, and purification of an extracellular protease produced by Aspergillus sp. (SIS 22/UCP 1287) under submerged fermentation. Enzyme extraction was coupled directly to fermentation using a polyethylene glycol (PEG)–phosphate aqueous two-phase system (ATPS), aiming to enhance recovery while preserving enzymatic activity. The effects of PEG molecular weight, polymer and phosphate concentrations, and pH on enzyme partitioning were systematically investigated through a full factorial experimental design. Low-molecular-weight PEG and near-neutral pH conditions significantly favored enzyme migration to the PEG-rich phase. Under optimized conditions (15% PEG 3500, 20% phosphate, pH 7.0), the ATPS achieved a partition coefficient of 65.55, enzyme recovery of 209%, and a purification factor of 1.64. Subsequent purification by DEAE–Sephadex ion-exchange chromatography yielded a tenfold increase in specific activity, with optimal elution at 0.5 M NaCl. SDS–PAGE analysis confirmed the homogeneity of the purified protease, revealing a single band at approximately 59 kDa. Overall, the proposed integrated ATPS–chromatography strategy represents a robust, scalable, and environmentally friendly platform that significantly simplifies downstream processing while maintaining high enzyme activity, highlighting its potential for industrial and biotechnological applications.