The improvement of stability, activity, and substrate promiscuity of glycerol dehydrogenase substituted by divalent metal ions
The improvement of stability, activity, and substrate promiscuity of glycerol dehydrogenase substituted by divalent metal ions
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DOI：
10.1007/s12257-013-0125-7
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发表时间：
2013-08
影响因子：
3.2
通讯作者：
Shizhen Wang;Jing Wang;Xiaofen Zhou;Yingxia Guo;B. Fang
中科院分区：
文献类型：
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作者：
Shizhen Wang;Jing Wang;Xiaofen Zhou;Yingxia Guo;B. Fang
肺炎克雷伯氏菌甘油脱氢酶催化锌离子的取代。通过二价金属离子Mn 2+和Mg 2+，能够改善活性、底物混杂性和稳定性。与天然GDH相比，Mn-GDH和Mg-GDH的活性提高了数倍。取代的GDH对非天然底物，4-氯乙酰乙酸，3-氯乙酰基吡啶，对氯苯乙酮，苯乙酮的活性比天然GDH高30倍。在60和70°C下，锰取代使GDH的半衰期增加了6倍。两组分一级失活模型能较好地拟合非线性热失活曲线。结合动力学和热力学分析，对金属离子在热稳定性增强中的作用机理进行了进一步的研究。失活的热力学参数、焓、熵和吉布斯自由能表明Mn-GDH是熵稳定的，并阐明了Mn-GDH失活的机理。
The substitution of the catalytic zinc ion of glycerol dehydrogenase (GDH) fromKlebsiella pneumoniasp. by divalent metal ions, Mn2+and Mg2+, enabled improvements of activity, substrate promiscuity and stability. The activity of Mn-GDH and Mg-GDH improved several folds in comparison to the native GDH. The activity of substituted GDH towards non-natural substrates, 4-chloroacetoacetate, 3-chloroacetylpyridine,p-chloroacetophenone, and acetophenone was 30 folds higher than native GDH. Manganese substitution increased the half-life of GDH by 6 folds at 60 and 70°C. The two-fraction first order inactivation models fitted the nonlinear thermal inactivation curves well. Combined with the kinetic and thermodynamic analysis, further mechanistic insights to the metal ion roles in thermostability enhancements were studied. The thermodynamic parameters of inactivation, enthalpy, entropy and the Gibbs free energy indicated that Mn-GDH was stabilized entropically and elucidated the mechanisms of enzyme inactivation.