CaXMT和TCS1突变体基因串联共表达及体外酶活检测分析

咖啡碱和可可碱是茶叶生物碱的主要组分,且咖啡碱是茶叶重要的滋味物质,随着咖啡碱在食品和药物领域的应用愈发广泛,咖啡碱的生物合成成为新的研究热点.目前市场上的咖啡碱主要靠化学合成,为了探索其生物合成途径,该研究将咖啡黄嘌呤核苷甲基转移酶(coffee xanthosine methyltransferase,CaXMT)基因和茶树咖啡碱合成酶(tea caffeine synthase,TCS1)基因的4个突变体分别串联至同一大肠杆菌表达载体pMAL-c5X,诱导融合蛋白共表达,并进行SDS-PAGE凝胶电泳分析.结果表明:目的蛋白成功表达后,应用超声破碎法制备含有目的蛋白的粗酶液,添加底物黄嘌呤核苷(xanthosine,XR)和甲基供体S-腺苷甲硫氨酸(S-adenosyl-L-methionine,SAM)进行体外酶促反应,将反应产物进行高效液相色谱检测.检测结果显示,pMAL-CaXMT-TM2/3/4的体外酶促反应产物仅有可可碱生成,均未见咖啡碱生成.该研究结果为构建生物合成咖啡碱和可可碱的串联共表达载体奠定了基础,也为进一步研究生物合成咖啡碱和可可碱提供了新思路.

Tandem coexpression of CaXMT and mutants of TCS1 and analysis of enzyme activity in vitro

Caffeine and theobromine as the major component of alkaloids in tea, while caffeine is the important taste compound. As the application of caffeine increased extensively in the fields of food and medicine, caffeine biosynthesis becomes a new hotspot. Nowadays, exploring the method of caffeine biosynthesis is of great significance, when caffeine synthesis mainly relies on chemical synthesis on the market. This research concatenated coffee xanthosine methyltransferase gene(CaXMT)and four mutants of tea caffeine synthase gene(TCS1)respectively in the same expression vector pMAL-c5X, then induced coexpression of the fusion protein, which was analyzed by SDS-PAGE gel electrophoresis, and chromatography were added into the crude enzyme solution by sonication methionine. At last, in vitro enzymatic reaction products were detected by high performance liquid. The results showed that only theobromine generated in the products of pMAL-CaXMT-TM2/3/4 without caffeine. This research provides the information for establishing tandem gene coexpression vector which was used for the biosynthesis of caffeine and theobromine and new ideas to study on caffeine biosynthesis.