A carboxymethyl cellulase from the yeast Cryptococcus gattii WM276: Expression, purification and characterisation.

Autor: Moodley D; School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, South Africa., Botes A; School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, South Africa. Electronic address: angela.botes@wits.ac.za.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2025 Jan; Vol. 225, pp. 106594. Date of Electronic Publication: 2024 Aug 26.
DOI: 10.1016/j.pep.2024.106594
Abstrakt: Cryptococcus gattii and its medical implications have been extensively studied. There is, however, a significant knowledge gap regarding cryptococcal survival in its environmental niche, namely woody material, which is glaring given that infection is linked to environmental populations. A gene from C. gattii (WM276), the predominant global molecular type (VGI), has been sequenced and annotated as a putative cellulase. It is therefore, of both medical and industrial intertest to delineate the structure and function of this enzyme. A homology model of the enzyme was constructed as a fusion protein to a maltose binding protein (MBP). The CGB_E4160W gene was overexpressed as an MBP fusion enzyme in Escherichia coli T7 cells and purified to homogeneity using amylose affinity chromatography. The structural and functional character of the enzyme was investigated using fluorescence spectroscopy and enzyme activity assays, respectively. The optimal enzyme pH and temperature were found to be 6.0 and 50 °C, respectively, with an optimal salt concentration of 500 mM. Secondary structure analysis using Far-UV CD reveals that the MBP fusion protein is primarily α-helical with some β-sheets. Intrinsic tryptophan fluorescence illustrates that the MBP-cellulase undergoes a conformational change in the presence of its substrate, CMC-Na + . The thermotolerant and halotolerant nature of this particular cellulase, makes it useful for industrial applications, and adds to our understanding of the pathogen's environmental physiology.
Competing Interests: Declaration of competing interest The authors have no competing interests to declare that are relevant to the content of this article.
(Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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