Conversion of xylose from renewable lignocellulosic source into value added products such as xylitol has gained importance with increasing global market for xylitol, which is being applied in health and food industries. Enzymatic synthesis of xylitol employing xylose reductases has several advantages over chemical and microbial methods. Xylose reductase from Debaryomyces nepalensis NCYC 3413 (DnXR) a potential candidate for xylitol synthesis industrially important properties such as halotolerance, higher turnover number, specific activity and better tolerance towards inhibitors generated during biomass pre-treatment. Since stability of enzymes is one of the factors that determine the economic feasibility of industrial enzymatic processes, the current study was undertaken.

Stability analysis was carried out in terms of deactivation kinetics, estimation of thermodynamic parameters, secondary and tertiary structural analysis through circular dichroism and tryptophan fluorescence. Though the activity was maximum at 55 ℃ and pH 6.5-7.0, the enzyme lost the function in a few minutes at 55 ℃. The gradual disruption of both secondary and tertiary structure of DnXR were responsible for enzyme denaturation beyond 40 ℃. DnXR showed a half-life of 5 h at 40 ℃ with melting temperature of 49 ℃. Since the melting temperature was less than the temperature of maximum activity, the protective role of substrate and co-substrate was anticipated. In the presence of NADPH (co-substrate), half-life time and melting temperature was improved. The stabilization of enzyme was brought about by conformational changes upon binding of cofactor to enzyme.

pH 6.5 to 7.0 yielded maximum activity whereas at pH 7.0 enzyme was most stable with half-life time of 5 h at 40 ℃. At extreme pH the enzyme was highly unstable with half-life time of 2 min and 10 min at pH 5.0 and 10.0 respectively. Disruption of secondary and tertiary structure was responsible for enzyme destabilization in acidic pH whereas the loss of tertiary structure alone was responsible for functional loss of enzyme in the alkaline pH. Enzyme stability was improved by employing osmolytes and ionic liquids (ILs). Among the osmolytes – trehalose, sucrose, glycerol and sorbitol – sorbitol was found to be the best stabilizer of DnXR. All the osmolytes improved the DnXR stability at extreme pH by protecting the secondary and tertiary structures of the enzyme. Sorbitol improved the half-life of DnXR by 9-folds at pH 5.0 and by 11-folds at pH 10.0. Tetramethylguanidium ([TMG]) based and Diazobicyclo[5.4.0]undec-7ene-ium ([DBU]) based ILs found to improve the DnXR activity and stability among the eight ILs tested. [DBU][Acetate] and [TMG][Butanoate] improved enzyme activity at pH 10.0 whereas [TMG][Acetate] enhanced the half-life time by 10-folds at pH 10.0.

Statistical studies yield empirical equations and help in developing efficient processes. Since the separate optimization of pH and temperature for maximum activity and stability yielded conditions which varied significantly and were deleterious for each other, simultaneous optimization was attempted. Simultaneous optimization of activity and stability resulted in optimum conditions of pH 7.1 and temperature 27 ℃, where values of activity and half-life time were moderate.

Publications:

Shwethashree Malla and Sathyanarayana N Gummadi (2018) Thermal stability of xylose reductase from Debaryomyces nepalensis NCYC 3413: deactivation kinetics and structural studies, Process Biochemistry, 67:71-79.

Shwethashree Malla and Sathyanarayana N Gummadi (2020), Counteraction of osmolytes on pH-induced unfolding of xylose reductase from Debaryomyces nepalensis NCYC 3413, European Biophysics Journal, 49:267–277.

Shwethashree Malla and Sathyanarayana N Gummadi (2020), Simultaneous optimization of activity and stability of xylose reductase from Debaryomyces nepalensis NCYC 3413 using statistical experimental design, Protein and Peptide Letters 27:1.

Shwethashree M, Jisha K J, Ramesh L Gardas, Sathyanarayana N. Gummadi (2021), Diazobicyclo[5.4.0]undec-7ene-ium and tetramethyl guanidium based ionic liquids enhanced thermal stability of xylose reductase at extreme pH through specific ion effect, Journal of Molecular Liquids, 328: 115394.

Publication not related to thesis:

Harshiny Muthukamar, Shwethashree Malla, Manickam Matheswaran, Sathyanarayana N Gummadi (2020), Immobilization of xylose reductase enzyme on cysteine-functionalized Murraya koenigii mediated magnetite nanoparticles, Materials Letters, 261: 127125.