Dietetics & Nutrition Technology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur 176061, Himachal Pradesh, India.
Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar 143005, Punjab, India.
J Agric Food Chem. 2023 Apr 12;71(14):5592-5599. doi: 10.1021/acs.jafc.3c00295. Epub 2023 Mar 31.
An extracellular γ-glutamyl transpeptidase (GGT) produced from IHB B1644 was purified to homogeneity employing ion-exchange chromatography. GGT comprised two subunits of 40 and 22 kDa determined by SDS-PAGE. The maximum enzyme activity was optimal at pH 9 and 37 °C. The purified enzyme was stable from pH 5-10 and <50 °C. Steady-state kinetic studies revealed a value of 0.538 mM against γ-GpNA. For substrate specificity, GGT showed highest affinity for l-methionine. The inhibitors' effect demonstrated that serine or threonine and tryptophan residues are essential for enzyme activity. l-Theanine production was optimized by employing a one-variable-at-a-time approach with 60-65% conversion rate. The final reaction consisted of 20 mM l-glutamine, 200 mM ethylamine hydrochloride, and 10 U mL enzyme concentration at 37 °C in Tris-Cl (50 mM, pH 9) for 5 h. l-Theanine was purified using a Dowex 50W X 8 hydrogen form resin and confirmed by HPLC and H NMR spectroscopies.
IHB B1644 产生的细胞外 γ-谷氨酰转肽酶（GGT）经离子交换层析纯化至均一性。GGT 通过 SDS-PAGE 确定由两个 40 和 22 kDa 的亚基组成。最大酶活在 pH 9 和 37°C 时最佳。纯化酶在 pH 5-10 和 <50°C 下稳定。稳态动力学研究表明，对 γ-GpNA 的 值为 0.538 mM。对于底物特异性，GGT 对 l-蛋氨酸表现出最高的亲和力。抑制剂的作用表明丝氨酸或苏氨酸和色氨酸残基对酶活性至关重要。采用单变量法优化 l-茶氨酸的生产，转化率为 60-65%。最终反应在 37°C 下于 Tris-Cl（50 mM，pH 9）中进行 5 小时，反应物为 20 mM l-谷氨酰胺、200 mM 乙基胺盐酸盐和 10 U mL 酶浓度。l-茶氨酸使用 Dowex 50W X 8 氢型树脂进行纯化，并通过 HPLC 和 H NMR 光谱进行确认。
