单 位：
安徽农业大学茶树生物学与资源利用国家重点实验室
关键词：
黑茶加工;咖啡碱;鸟嘌呤脱氨酶;黄嘌呤;生物合成;原核表达
摘 要：
与植物体内合成路径不同,微生物体内合成咖啡碱存在一条以黄嘌呤为底物,利用鸟嘌呤脱氨酶催化鸟嘌呤生成黄嘌呤有效合成咖啡碱的新途径。为克隆鸟嘌呤脱氨酶的基因,构建可高效合成黄嘌呤的原核表达载体并对外源蛋白活性进行检测,分别以酿酒酵母和大肠杆菌为研究材料,根据Gen Bank中酿酒酵母和大肠杆菌中鸟嘌呤脱氨酶基因gud1和egud序列设计引物,聚合酶链式反应特异扩增其基因片段,将目的基因连接至p MAL-c5X载体,转入大肠杆菌BL21(DE3)中诱导蛋白表达,并用高效液相色谱法鉴定其目的蛋白的催化活性。结果表明重组载体p MAL-gud1、p MAL-egud均可用来合成黄嘌呤,且GUD1比EGUD合成黄嘌呤的效率更高。研究结果将进一步丰富黑茶加工技术理论,同时为体外构建高效咖啡碱生物工程菌提供理论支持。
译 名：
Cloning, Prokaryotic Expression and Functional Identification of Guanine Deaminase Genes from Saccharomyces cerevisiae and Escherichia coli
作 者：
SUN Ying;PAN Si'an;LI Mengmeng;DENG Weiwei;ZHANG Zhengzhu;State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University;
关键词：
dark tea processing;;caffeine;;guanine deaminase;;xanthine;;biosynthesis;;prokaryotic expression
摘 要：
A novel pathway for the biosynthesis of caffeine with xanthine as substrate has been found in microbes, which is an effective way to improve the efficiency of caffeine synthesis from xanthine produced from guanine catalyzed by guanine deaminase. This study aimed to clone the guanine deaminase gene and to construct a prokaryotic expression system to synthesize xanthine efficiently. The two guanine deaminase genes gud1 and egud were amplified from Saccharomyces cerevisiae and Escherichia coli by PCR, respectively. The target fragments were inserted into the p MAL-c5 X vector and then the recombinant plasmid transformed into E. coli BL21(DE3) to induce protein expression. The expressed products were determined by high performance liquid chromatography(HPLC). Both the recombinant vectors p MAL-gud1 and p MAL-egud had the capability of synthesizing xanthine, and the efficiency of xanthine synthesis with GUD1 was higher as compared to EGUD. This study can enrich the theory of black tea processing technology and provide a theoretical foundation for constructing engineered strains that are capable of producing caffeine.
