Increasing the thermostability of glutamate decarboxylase from Lactobacillus brevis by introducing proline.

Hui Fang; Changjiang LÜ; Yujiao Hua; Sheng HU; Weirui Zhao; Wenji Fang; Kui Song; Jun Huang; Lehe Mei

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Increasing the thermostability of glutamate decarboxylase from Lactobacillus brevis by introducing proline.

Author: Hui FANG ¹ ; Changjiang LÜ ¹ ; Yujiao HUA ¹ ; Sheng HU ² ; Weirui ZHAO ² ; Wenji FANG ¹ ; Kui SONG ¹ ; Jun HUANG ¹ ; Lehe MEI ²
Author Information

1. School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, Zhejiang, China.
2. Department of Biological and Pharmaceutical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, Zhejiang, China.
Publication Type:Journal Article
Keywords: Lactobacillus brevis; glutamate decarboxylase; molecular dynamics; proline; thermostability; γ-aminobutyric acid
MeSH: Glutamate Decarboxylase; Glutamic Acid; Lactobacillus brevis; Molecular Dynamics Simulation; Proline
From: Chinese Journal of Biotechnology 2019;35(4):636-646
CountryChina
Language:Chinese
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 (T₅₀ ¹⁵) 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.