Department of Food Science, College of Agriculture and Health, Tunghai University, No. 1727, Sec. 4, Taiwan Boulevard, Xitun District, Taichung 40704, Taiwan.
Department of Food Science, College of Agriculture and Health, Tunghai University, No. 1727, Sec. 4, Taiwan Boulevard, Xitun District, Taichung 40704, Taiwan.
Int J Biol Macromol. 2024 Oct;278(Pt 4):135046. doi: 10.1016/j.ijbiomac.2024.135046. Epub 2024 Aug 23.
This study harnesses glutamate decarboxylase (GAD) from Yarrowia lipolytica to improve the biosynthesis of γ-aminobutyric acid (GABA), focusing on boosting the enzyme's catalytic efficiency and stability by immobilizing it on nanofibrous membranes. Through recombinant DNA techniques, two GAD genes, YlGAD1 and YlGAD2, were cloned from Yarrowia lipolytica and then expressed in Escherichia coli. Compared to their soluble forms, the immobilized enzymes exhibited significant improvements in thermal and pH stability and increased resistance to chemical denaturants. The immobilization notably enhanced substrate affinity, as evidenced by reduced K values and increased k values, indicating heightened catalytic efficiency. Additionally, the immobilized YlGAD1 and YlGAD2 enzymes showed substantial reusability, maintaining 50% and 40% of their activity, respectively, after six consecutive cycles. These results underscore the feasibility of employing immobilized YlGAD enzymes for cost-effective and environmentally sustainable GABA production. This investigation not only affirms the utility of YlGADs in GABA synthesis but also underscores the advantages of enzyme immobilization in industrial settings, paving the way for scalable biotechnological processes.
本研究利用解脂耶氏酵母中的谷氨酸脱羧酶（GAD）来提高γ-氨基丁酸（GABA）的生物合成效率，重点通过将其固定在纳米纤维膜上来提高酶的催化效率和稳定性。通过重组 DNA 技术，从解脂耶氏酵母中克隆了两个 GAD 基因 YlGAD1 和 YlGAD2，然后在大肠杆菌中表达。与它们的可溶性形式相比，固定化酶在热稳定性和 pH 稳定性方面有显著提高，并且对化学变性剂的抵抗力增强。固定化显著提高了底物亲和力，表现为 K 值降低和 k 值增加，表明催化效率提高。此外，固定化的 YlGAD1 和 YlGAD2 酶具有很高的可重复使用性，在连续六次循环后，其活性分别保持在 50%和 40%。这些结果证实了固定化 YlGAD 酶在经济高效和环境可持续 GABA 生产中的可行性。这项研究不仅证实了 YlGAD 在 GABA 合成中的实用性，还强调了酶固定化在工业环境中的优势，为可扩展的生物技术过程铺平了道路。
