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Abstract：
γ-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 ( http://cbs-kazlab.oit.umn.edu/ ), 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 °C increase in semi-inactivation temperature (T5015), a 1.7-fold improvement of half-life (t1/2) at 55 °C, and a 1.2-fold improvement of t1/2 at 37 °C, respectively, while its catalytic efficiency (kcat/Km) 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 kcat/Km 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.
摘要：
γ-氨基丁酸酯(GABA)是通过谷氨酸脱羧酶(GAD)使L-谷氨酸脱羧合成的重要生物活性化合物。在这项研究中,通过共有诱变构建来自短乳杆菌的GAD的稳定变体。使用共识查找器(http://cbs-kazlab。oit.umn.edu/)，鉴定了同源家族成员中具有最普遍氨基酸(超过60%阈值)的八个位置。随后，使用定点诱变将这八个残基分别突变以匹配共有序列。与野生型相比,T383K变体在单个变体中显示出最大的热稳定性变化，半失活温度（T5015）升高3.0°C，在55°C下半衰期（t1/2）提高1.7倍，在37°C时t1/2提高了1.2倍，分别,而其催化效率(kcat/Km)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进行了位点饱和突变.值得注意的是,突变体T383V和T383G的热稳定性和kcat/Km比野生型增加。本研究不仅强调了一致诱变对提高GAD热稳定性的价值，而且为研究其他酶的热稳定性提供了有力的指导。
