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.

中文翻译：

---

渗透液对pH诱导的尼泊尔脱皮芽孢杆菌NCYC 3413中木糖还原酶展开的反作用。

研究了尼泊尔Debaryomyces nepalensis NCYC 3413木糖还原酶（一种从大肠杆菌Rosetta细胞重组表达并纯化的同型二聚酶）在5.0至10.0的不同pH值下的稳定性。通过分析重组酶在40°C下随时间的残留活性研究了在不同pH值下的失活动力学，而使用圆二色性和差示扫描量热法研究了构象变化和稳定性依赖性。四个渗透液即。研究了甘油，蔗糖，海藻糖和山梨糖醇对中性和极端pH条件下酶的失活和解链温度的影响。发现该酶在pH 7.0时具有催化和结构稳定性，半衰期为250分钟，熔融温度为50°C。发现在二级和三级结构中的改变均引起酸性pH下的酶失活，而在碱性pH下失活速率的增加归因于三级结构随时间的变化。估计的热力学参数还表明，酶的稳定性在中性pH下最高，ΔH为348 kcal / mol，ΔG40为9.53 kcal / mol。在极端pH下，所有四种渗透液都能有效地将酶稳定性提高几倍，其中山梨糖醇最为有效，在pH 10.0时酶半衰期增加了11倍，在pH 5.0时酶半衰期增加了8倍。估计的热力学参数还表明，酶的稳定性在中性pH下最高，ΔH为348 kcal / mol，ΔG40为9.53 kcal / mol。在极端pH下，所有四种渗透液都能有效地将酶稳定性提高几倍，其中山梨糖醇最为有效，在pH 10.0时酶半衰期增加了11倍，在pH 5.0时酶半衰期增加了8倍。估计的热力学参数还表明，酶的稳定性在中性pH值最高，ΔH为348 kcal / mol，ΔG40为9.53 kcal / mol。在极端pH下，所有四种渗透液都能有效地将酶稳定性提高几倍，其中山梨糖醇最为有效，在pH 10.0时酶半衰期增加了11倍，在pH 5.0时酶半衰期增加了8倍。