Extraction of collagenolytic proteases from Aspergillus heteromorphus URM 0269 in an aqueous two-phase system for application in collagen hydrolysis

Abstract

Collagenolytic proteases produced by Aspergillus heteromorphus URM0269 were extracted using a PEG/sulfate aqueous two-phase system (ATPS). A 2³ factorial design was performed to analyze the independent variables: PEG molar mass (M_PEG), PEG concentration (C_PEG), and sulfate concentration (C_sulf). The extracted proteases were also evaluated for their optimum pH and stability at different pH levels (4.0 − 11.0) after 20 h of incubation. Collagen was extracted from mutton snapper (Lutjanus analis) skin using acetic acid (0.5 mol L⁻¹). The enzyme was preferentially partitioned to the PEG-rich phase (K > 1), whose highest purification factor and recovery (PF = 6.256 and Y = 404.432%) were obtained under specific conditions: M_PEG 8000 g.mol⁻¹, C_PEG 30%, C_sulf 10%. The ATPS extraction provided an enzymatic activity range of pH 7.0 − 11.0, exhibiting greater stability compared to the crude extract. Approximately 80% of protease activity was maintained after 20 hours of incubation at all analyzed pH levels, except pH 11.0. Collagen extraction from L. analis skin yielded 8.056%, and both crude extract samples and ATPS-derived samples successfully hydrolyzed the extracted collagen, reaching peak hydrolysis after 36 hours of treatment. These findings demonstrate the feasibility of extracting highly purified and active proteases capable of hydrolyzing L. analis collagen.

Keywords:

• Fish waste
• enzyme partitioning
• collagen extraction

Acknowledgements

The authors would like to thank the Graduate Program in Animal Bioscience (PGBA), the Coordination for the Improvement of Higher Education Personnel (CAPES) for the master’s scholarship (N. 88887.495378/2020-00), the National Council for Scientific and Technological Development (CNPq) for supporting this work, and the Foundation for Science and Technology of the State of Pernambuco (FACEPE, Brazil) for its financial support (APQ-0726-5.07/21). Tatiana Porto is grateful to National Council for Scientific and Technological Development (CNPq, Brazil) for the Research Productivity Scholarship (315249/2021-8).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The present work was funded by the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) scholarship number 88887.495378/2020-00, the National Council for Scientific and Technological Development (CNPq, Brazil) grant number 315249/2021-8, and the Foundation for Science and Technology of the State of Pernambuco (FACEPE, Brazil) (APQ-0726-5.07/21).