Applied Biochemistry and Biotechnology ( IF 3 ) Pub Date : 2020-03-03 , DOI: 10.1007/s12010-020-03283-0 Yujiao Hua 1 , Changjiang Lyu 1 , Chunyan Liu 1 , Hongpeng Wang 1 , Sheng Hu 2 , Weirui Zhao 2 , Jiaqi Mei 3 , Jun Huang 1 , Lehe Mei 2
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.
中文翻译:
通过共识诱变提高短乳杆菌谷氨酸脱羧酶的热稳定性。
γ-氨基丁酸酯(GABA)是一种重要的生物活性化合物,是通过谷氨酸脱羧酶(GAD)使L-谷氨酸脱羧而合成的。在本研究中,通过共有诱变构建了短乳杆菌GAD的稳定变体。使用共识发现器(http://cbs-kazlab.oit.umn.edu/),鉴定了同源家族成员中氨基酸含量最高(超过阈值60%)的八个位置。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,T383K变体在单个变体中表现出最大的热稳定性变化,半灭活温度升高了3.0°C(T 50 15),在55°C下的半衰期(t 1/2)改善了1.7倍,在37°C下的t 1/2改善了1.2倍,同时其催化效率(k cat / K m)减少了。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比野生型表现出热稳定性和k cat / K m的增加。这项研究不仅强调了共有诱变对于提高GAD的热稳定性的价值,而且为研究其他酶的热稳定性提供了有力的指导。