Improving the Thermostability of Glutamate Decarboxylase from Lactobacillus brevis by Consensus Mutagenesis
Improving the Thermostability of Glutamate Decarboxylase from Lactobacillus brevis by Consensus Mutagenesis
复制标题
通过一致性诱变提高短乳杆菌谷氨酸脱羧酶的热稳定性
DOI：
10.1007/s12010-020-03283-0
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发表时间：
2020-03-03
影响因子：
3
通讯作者：
Mei, Lehe
中科院分区：
文献类型：
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作者：
Hua, Yujiao;Lyu, Changjiang;Mei, Lehe
γ-氨基丁酸（GABA）是由谷氨酸脱羧酶（GAD）催化L-谷氨酸脱羧合成的一种重要生物活性物质。在这项研究中，从短乳杆菌的GAD的稳定的变体构建的共识诱变。使用共有序列（），鉴定了同源家族成员中具有最普遍氨基酸（超过60%阈值）的8个位置。随后，使用定点诱变将这八个残基单独突变以匹配共有序列。与野生型相比，T383 K变体在单一变体中显示出最大的热稳定性变化，半失活温度（T-50（15））增加3.0 ℃，55 ℃下半衰期（t（1/2））提高1.7倍，37 ℃下t（1/2）提高1.2倍，催化效率（k（cat）/K-m）降低。为了获得热稳定性和催化活性均得到改善的突变体，我们在T383处进行了位点饱和突变。突变体T383 V和T383 G的热稳定性和k（cat）/K-m均比野生型有所提高。该研究不仅强调了共识突变对于提高GAD热稳定性的价值，而且对研究其他酶的热稳定性具有强有力的指导作用。
gamma-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). In this study, stabilized variants of the GAD from Lactobacillus brevis were constructed by consensus mutagenesis. Using Consensus Finder (), eight positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these eight residues were individually mutated to match the consensus sequence using site-directed mutagenesis. Compared to the wild-type, T383K variant displayed the largest shift in thermostability among the single variants, with a 3.0 degrees C increase in semi-inactivation temperature (T-50(15)), a 1.7-fold improvement of half-life (t(1/2)) at 55 degrees C, and a 1.2-fold improvement of t(1/2) at 37 degrees C, respectively, while its catalytic efficiency (k(cat)/K-m) was reduced. To obtain the mutant with improvement in both thermostability and catalytic activity, we performed a site-saturation mutation at T383. Notably, mutants T383V and T383G exhibited an increasement in thermostability and k(cat)/K-m than that of wild-type. This study not only emphasizes the value of consensus mutagenesis for improving the thermostability of GAD but also sheds a powerful guidance to study the thermal stability of other enzymes.