Arai Shintaro, Suzuki Hideyuki
Division of Applied Biology, Kyoto Institute of Technology, Goshokaido-Cho, Matsugasaki, Sakyo-Ku, Kyoto, 606-8585, Japan.
AMB Express. 2023 Mar 4;13(1):27. doi: 10.1186/s13568-023-01528-9.
An Escherichia coli strain expressing γ-glutamyltranspeptidase on its extracellular surface using the Met1 to Arg232 fragment of YiaT of E. coli as an anchor protein was immobilized with alginate for repeated use. Measurement of γ-glutamyltranspeptidase activity of the immobilized cells was performed repeatedly at pH 8.73 and 37 °C for 10 days using γ-glutamyl-p-nitroanilide in the presence of 100 mM CaCl and 3% NaCl with and without glycylglycine. Even after the 10th day, the enzyme activity did not decrease from the initial levels. The production of γ-glutamylglutamine from glutamine using the immobilized cells was performed repeatedly at pH 10.5 and 37 °C for 10 days in the presence of 250 mM glutamine, 100 mM CaCl, and 3% NaCl. Sixty-four % of glutamine was converted to γ-glutamylglutamine in the first cycle. While repeating the production 10 times, the surface of the beads gradually became covered with white precipitate, and the conversion efficiency gradually decreased, but 72% of the initial value still remained even at the 10th measurement.
一株利用大肠杆菌YiaT的Met1至Arg232片段作为锚定蛋白在细胞外表面表达γ-谷氨酰转肽酶的大肠杆菌菌株，用海藻酸盐固定化以重复使用。在100 mM CaCl和3% NaCl存在及不存在甘氨酰甘氨酸的情况下，于pH 8.73和37°C使用γ-谷氨酰对硝基苯胺对固定化细胞的γ-谷氨酰转肽酶活性进行了10天的重复测定。即使在第10天后，酶活性也未从初始水平下降。在250 mM谷氨酰胺、100 mM CaCl和3% NaCl存在的情况下，于pH 10.5和37°C使用固定化细胞从谷氨酰胺生产γ-谷氨酰谷氨酰胺进行了10天的重复实验。在第一个循环中，64%的谷氨酰胺转化为γ-谷氨酰谷氨酰胺。在重复生产10次的过程中，珠子表面逐渐被白色沉淀覆盖，转化效率逐渐降低，但即使在第10次测量时，仍保留了初始值的72%。
