Abstract：
L-theanine is an amino acid with a unique flavor and many therapeutic effects. Its enzymatic synthesis has been actively studied and γ-Glutamylmethylamide synthetase (GMAS) is one of the promising enzymes in the biological synthesis of theanine. However, the theanine biosynthetic pathway with GMAS is highly ATP-dependent and the supply of external ATP was needed to achieve high concentration of theanine production. As a result, this study aimed to investigate polyphosphate kinase 2 (PPK2) as ATP regeneration system with hexametaphosphate. Furthermore, the alginate entrapment method was employed to immobilize whole cells containing both gmas and ppk2 together resulting in enhanced reusability of the theanine production system with reduced supply of ATP. After immobilization, theanine production was increased to 239 mM (41.6 g/L) with a conversion rate of 79.7% using 15 mM ATP and the reusability was enhanced, maintaining a 100% conversion rate up to the fifth cycles and 60% of conversion up to eighth cycles. It could increase long-term storage property for future uses up to 35 days with 75% activity of initial activity. Overall, immobilization of both production and cofactor regeneration system could increase the stability and reusability of theanine production system.
摘要：
L-茶氨酸是一种具有独特风味和许多治疗作用的氨基酸。其酶法合成已被积极研究，γ-谷氨酰甲基酰胺合成酶（GMAS）是茶氨酸生物合成中很有前途的酶之一。然而,具有GMAS的茶氨酸生物合成途径是高度ATP依赖性的,并且需要外部ATP的供应来实现高浓度的茶氨酸生产。因此,本研究旨在研究聚磷酸激酶2（PPK2）作为六偏磷酸盐的ATP再生体系。此外，采用藻酸盐包封方法将含有gmas和ppk2的全细胞固定在一起，从而提高了茶氨酸生产系统的可重用性，同时减少了ATP的供应。固定后，茶氨酸产量增加到239mM（41.6g/L），使用15mMATP的转化率为79.7％，可重用性增强，保持100%的转化率直至第五循环和60%的转化率直至第八循环。它可以增加长期储存性能，用于未来使用长达35天，具有初始活性的75％活性。总的来说,生产和辅因子再生系统的固定化可以提高茶氨酸生产系统的稳定性和可重用性。
