Abstract
The stability of Debaryomyces nepalensis NCYC 3413 xylose reductase, a homodimeric enzyme recombinantly expressed and purified from E. coli Rosetta cells, was studied at different pH ranging from 5.0 to 10.0. Deactivation kinetics at different pH were studied by analyzing residual activity of the recombinant enzyme over time at 40 °C whereas conformational changes and stability dependence were investigated by using circular dichroism and differential scanning calorimetry. Four osmolytes viz. glycerol, sucrose, trehalose and sorbitol were explored for their effect on the deactivation and melting temperatures of the enzyme under neutral and extreme pH conditions. The enzyme was found to be catalytically and structurally stable at pH 7.0 with half-life of 250 min and a melting temperature of 50 °C. It was found that alteration in both secondary and tertiary structures caused enzyme deactivation in acidic pH while increased deactivation rates at alkaline pH was attributed to the variation of tertiary structure over time. Estimated thermodynamic parameters also showed that the enzyme stability was highest at neutral pH with ΔH of 348 kcal/mole and ΔG40 of 9.53 kcal/mole. All four osmolytes were effective in enhancing enzyme stability by several folds at extreme pH with sorbitol being the most efficient, which increased enzyme half-life by 11-fold at pH 10.0 and 8-fold at pH 5.0.
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Authors acknowledge Department of Biotechnology, Government of India for funding, and IIT Madras for the research facilities as well as DST-FIST facility for CD and DSC instruments. SM acknowledges MHRD, Government of India for the fellowship. Authors acknowledge Dr. Swati Dash for her kind suggestions in improving manuscript.
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SNG devised and designed the studies and SM performed the experiments. SNG framed the outline of manuscript and suggested corrections whereas SM prepared the manuscript. Both the authors read and agreed the complete manuscript.
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Malla, S., Gummadi, S.N. Counteraction of osmolytes on pH-induced unfolding of xylose reductase from Debaryomyces nepalensis NCYC 3413. Eur Biophys J 49, 267–277 (2020). https://doi.org/10.1007/s00249-020-01432-1
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DOI: https://doi.org/10.1007/s00249-020-01432-1