Development of a fluorescence-based excipient screening for improved stability and shelf-life of recombinant chitin deacetylase.

Autor: Pawaskar GM; Department of Biotechnology, Manipal Institute of Technology, Manipal Academy of Higher Education (MAHE), Manipal, 576104, India., Raval R; Department of Biotechnology, Manipal Institute of Technology, Manipal Academy of Higher Education (MAHE), Manipal, 576104, India.
Jazyk: angličtina
Zdroj: Biochemistry and biophysics reports [Biochem Biophys Rep] 2024 Apr 27; Vol. 38, pp. 101718. Date of Electronic Publication: 2024 Apr 27 (Print Publication: 2024).
DOI: 10.1016/j.bbrep.2024.101718
Abstrakt: Chitin deacetylase (CDA) modifies chitin into chitosan by removing acetyl groups, but its inherent instability poses a challenge for successful crystallisation. Despite limited successes in crystallizing CDAs, prior attempts with recombinant chitin deacetylase ( Ba CDA) failed due to poor stability. To address this, we propose an enzyme buffer formulation as a cost-effective strategy to enhance stability, prolong shelf life, and increase the likelihood of crystallisation. Utilizing the high-throughput screening technique FTSA, we developed a screening method correlating Ba CDA stability with its activity. The optimised formulation comprises 50 mM Tris-HCl buffer pH 7, 1 M NaCl, 20 % glycerol, and 1 mM Mg 2+ as excipients. This formulation significantly improves Ba CDA's thermostability (140.47 % increase) and enzyme activity (2.9-fold enhancement). Ba CDA remains stable in the formulated buffer at -20 °C and -80 °C for 30 days and at 4 °C for 15 days. The current study has designed a high-throughput screening method approach to assess the stability of CDA enzyme formulations. The results of this study could contribute to the exploration of formulation elements that enhance the structural stability of CDA, thereby facilitating investigations into the enzyme's structure-function relationships.
Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(© 2024 The Authors.)
Databáze: MEDLINE