PDF(Pubmed)
Abstract：
L-Theanine, found in green tea leaves has been shown to positively affect immunity and relaxation in humans. There have been many attempts to produce L-theanine through enzymatic synthesis to overcome the limitations of traditional methods. Among the many genes coding for enzymes in the L-theanine biosynthesis, glutamylmethylamide synthetase (GMAS) exhibits the greatest possibility of producing large amounts of production. Thus, GMAS from Methylovorus mays No. 9 was overexpressed in several strains including vectors with different copy numbers. BW25113(DE3) cells containing the pET24ma::gmas was selected for strains. The optimal temperature, pH, and metal ion concentration were 50°C, 7, and 5 mM MnCl2, respectively. Additionally, ATP was found to be an important factor for producing high concentration of L-theanine so several strains were tested during the reaction for ATP regeneration. Bakers yeast was found to decrease the demand for ATP most effectively. Addition of potassium phosphate source was demonstrated by producing 4-fold higher L-theanine. To enhance the conversion yield, GMAS was additionally overexpressed in the system. A maximum of 198 mM L-theanine was produced with 16.5 mmol/l/h productivity. The whole-cell reaction involving GMAS has greatest potential for scale-up production of L-theanine.
摘要：
L-茶氨酸,绿茶叶中的发现已被证明对人类的免疫力和放松有积极影响。已经有许多尝试通过酶合成来生产L-茶氨酸以克服传统方法的局限性。在许多编码L-茶氨酸生物合成酶的基因中，谷氨酰甲基酰胺合成酶（GMAS）显示出产生大量生产的最大可能性。因此，来自Methylovorus的GMAS编号9在包括具有不同拷贝数的载体的几种菌株中过表达。选择含有pET24ma::gmas的BW25113(DE3)细胞作为菌株。最佳温度,pH值,金属离子浓度为50℃,7，和5mMMnCl2，分别。此外,发现ATP是产生高浓度L-茶氨酸的重要因素，因此在ATP再生反应过程中测试了几种菌株。发现面包酵母最有效地减少对ATP的需求。通过产生4倍高的L-茶氨酸证明了磷酸钾源的添加。为了提高转化率，GMAS在系统中另外过表达。以16.5mmol/l/h的生产率产生最多198mML-茶氨酸。涉及GMAS的全细胞反应对于扩大L-茶氨酸的生产具有最大的潜力。
