利用脯氨酸效应提高短乳杆菌谷氨酸脱羧酶的热稳定性
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国家自然科学基金 (Nos. 31470793, 31670804, 31240054),浙江省自然科学基金 (Nos. LY16B060008,LZ13B060002) 资助。


Increasing the thermostability of glutamate decarboxylase from Lactobacillus brevis by introducing proline
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National Natural Science Foundation of China (Nos. 31470793, 31670804, 31240054), Natural Science Foundation of Zhejiang Province (Nos. LY16B060008, LZ13B060002).

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    摘要:

    以磷酸吡哆醛为辅酶的谷氨酸脱羧酶 (Glutamate decarboxylase,GAD),能专一、不可逆地催化L-谷氨酸脱去α-羧基生成γ-氨基丁酸。为了提高GAD热稳定性为目标,本研究通过与嗜热古细菌Thermococcus kodakarensis中GAD氨基酸序列的比对及引入脯氨酸策略,最终在短乳杆菌Lactobacillus brevis CGMCC No.1306 的GAD突变体中筛选得到热稳定性提高的突变酶G364P。结果显示,突变酶G364P在55 ℃的半衰期以及半失活温度分别比野生酶提高19.4 min和5.3 ℃,并且突变酶G364P的催化效率与野生酶相比没有明显变化。此外,利用分子动力学模拟来验证突变对蛋白质热稳定性的影响,突变酶G364P的均方根偏差 (Root mean square deviation,RMSD) 以及含G364的loop区域均方根涨落 (Root mean square fluctuation,RMSF) 均比野生酶低,引入脯氨酸增加了364位氨基酸与相邻氨基酸的疏水相互作用。文中通过引入脯氨酸成功提高了L. brevis中GAD的热稳定性,同时也为其他酶热稳定性的理性设计提供了方法学指导。

    Abstract:

    Glutamate decarboxylase, a unique pyridoxal 5′-phosphate-dependent enzyme, catalyzes α-decarboxylation of L-glutamate to γ-aminobutyrate. However, glutamate decarboxylase from different sources has the common problem of poor thermostability that affects its application in industry. In this study, proline was introduced at 13 different positions in glutamate decarboxylase by using the design strategy of homologous sequence alignment between Thermococcus kodakarensis and Lactobacillus brevis CGMCC No.1306. A mutant enzyme G364P with higher thermostability was obtained. Compared to the wild type, thermostability of the mutant G364P was significantly improved, the half-life time (t1/2) at 55 °C and the semi-inactivation temperature (T5015) of the mutant G364P increased 19.4 min and 5.3 °C, respectively, while kcat/Km of the mutant enzyme remained nearly unchanged. Further analysis of their thermostability by molecular dynamics simulations were performed. The root mean square deviation of G364P and root mean square fluctuation in the loop region including G364 were lower than the wild type at 313 K for 10 ns, and G364P increased one hydrophobic interaction in the loop region. It proves that mutation of flexible 364-Gly to rigid proline endows glutamate decarboxylase with enhanced thermostability.

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方卉,吕常江,花雨娇,胡升,赵伟睿,方文姬,宋奎,黄俊,梅乐和. 利用脯氨酸效应提高短乳杆菌谷氨酸脱羧酶的热稳定性[J]. 生物工程学报, 2019, 35(4): 636-646

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  • 收稿日期:2018-09-20
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  • 在线发布日期: 2019-04-18
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