Purification and characterization of a collagenase from Penicillium sp. UCP 1286 by polyethylene glycol-phosphate aqueous two-phase system.

Collagenases are proteolytic enzymes capable of degrading both native and denatured collagen, reported to be applied in industrial, medical and biotechnological sectors. Liquid-liquid extraction using aqueous two-phase system (ATPS) is one of the most promising bioseparation techniques, which can substitute difficult solid-liquid separation processes, offering many advantages over conventional methods including low-processing time, low-cost material and low-energy consumption. The collagenase produced by Penicillium sp. UCP 1286 showed a stronger affinity for the bottom salt-rich phase, where the highest levels of collagenolytic activity were observed at the center point runs, using 15.0% (w/w) PEG 3350 g/mol and 12.5% (w/w) phosphate salt at pH 7.0 and concentration. The enzyme was characterized by thermal stability, pH tolerance and effect of inhibitors, showing optimal collagenolytic activity at 37 °C and pH 9.0 and proved to be a serine protease. ATPS showed high efficiency in the collagenase purification, confirmed by a single band in SDS/PAGE, and can in fact be applied as a quick and inexpensive alternative method.

Application of the galactomannan gel from Cassia grandis seeds for biomedical purposes: Study of the incorporation of collagenases and their release profile.

The galactomannan-based gel from Cassia grandis seeds was used to incorporate Penicillium sp. UCP 1286 and commercial collagenases. Experiments were carried out according to a 23-full factorial design to identify the most significant parameters for the incorporation process. The pH of the incorporation solution (pHi), stirring time (t), and initial protein concentration in the crude extract (PCi) were selected as the three independent variables, and the efficiency of collagenase incorporation (E) and collagenolytic activity (CA) after 360 min as the responses. pHi and PCi showed positive statistically significant effects on E, while CA was positively influenced by pHi and t, but negatively by PCi. The fungi collagenase was released from the gel following a pseudo-Fickian behavior. Additionally, no <76 % of collagenase was efficiently incorporated into the gel retaining a high CA (32.5-69.8 U/mL). The obtained results for the commercial collagenase (E = 93.88 %, CA = 65.8 U/mL, and n = 0.10) demonstrated a pseudo-Fickian behavior similar to the fungi-collagenase. The results confirm the biotechnological potential of the gel as an efficient matrix for the incorporation of catalytic compounds; additionally, the incorporation of collagenases was achieved by retaining the proteases CA and releasing them in a controlled manner.