|a英文題名:Molecular Cloning and Expression of Caffeine Demethylase from Burkholderia Cepacia Complex
|a咖啡因(1,3,7-trimethylxanthine)廣泛存在於多種日常食品中，過量攝取對人體健康具不良影響，如誘發心血管疾病、造成胎兒畸形和降低生育率等，因此去除食品中的咖啡因成為一項重要的加工技術。本研究旨於自種植咖啡樹之土壤中篩選具分解咖啡因能力之菌株，而後選殖其咖啡因去甲基酵素(Caffeine demethylase，CDase)基因，並於Escherichia coli細胞中大量表現，期望此基因重組之咖啡因去甲基酵素可應用於食品加工業。首先，自咖啡豆培植土中篩選200株具降解咖啡因能力之菌株，利用High-performance liquid chromatography (HPLC)評估咖啡因降解活性後，挑選活性最佳之菌株進行菌種鑑定，鑑定結果顯示其為Burkholderia cepacia complex中的一員。以polymerase chain reaction選殖B. cepacia之咖啡因去甲基酵素基因，並將其重組於表現載體pTrcHis A，之後於E. coli TOP10中大量表現。結果顯示，B. cepacia之咖啡因降解活性為0.056 g/L/h，經基因定序後得知其咖啡因去甲基酵素基因含有973 bp，且其DNA定序與Takeuchi, K.和Koide, Y.在1996年所發表Pseudomonas putida之咖啡因去甲基酵素相似度為99 %。由SDS-PAGE得知基因重組之咖啡因去甲基酵素之蛋白質分子量約為41.58 kDa。Western blot和咖啡因降解試驗結果顯示，重組之咖啡因去甲基酵素為包含體(inclusion body)，不具酵素活性；但利用0.5 % SDS使重組之咖啡因去甲基酵素蛋白質變性，再以2 mM reduced glutathione和0.2 mM oxidized glutathione使之復性後，此重組酵素降解咖啡因的能力提升為0.67 mg/L/min。|uCaffeine (1,3,7-trimethylxanthine) is widely present in many foods, excessive intake may have adverse effects on human health. For example, cardiovascular disease, fetus malformation and reduction of fertility rate may cause by the consumption of high caffeine-containing foods. Hence, de-caffeine is an important food processing technique for improving consumers’ health. The aims of this study were to isolate caffeine degrading strains from the soil of coffee plantation area, to clone the caffeine demethylase (CDase) gene from the strains and over-express in Escherichia coli. The recombinant CDase could be used in the food industry for de-caffeine process. In this study, 200 strains were isolated from the soil and the caffeine degradation activities of the strains were assessed by High-performance liquid chromatography (HPLC). The strain with the highest ability to degrade caffeine was identified as Burkholderia cepacia complex. The caffeine demethylase gene from B. cepacia was cloned by polymerase chain reaction, recombined in expression vector (pTrcHis A) and over-expressed in Escherichia coli TOP10. The results showed that the caffeine degradation activity of B. cepacia was 0.056 g/L/h. DNA sequencing indicated that the CDase gene had 973 bp and showed the homology of 99 % with the CDase reported from Pseudomonas putida by Takeuchi, K. and Koide, Y. in 1996. Molecular weight of the recombinant CDase was about 41.58 kDa based on the measurement of SDS-PAGE. Western blot and caffeine degradation test results showed that the recombinant CDase was an inclusion body and showed no caffeine degradation activity. However, after the denaturation of the recombinant CDase with 0.5% SDS and the renaturation using 2 mM reduced glutathione and 0.2 mM oxidized glutathione, the refolded recombinant CDase showed caffeine degradation activity of 0.67 mg/L/min.
|aMolecular Cloning and Expression of Caffeine Demethylase from Burkholderia Cepacia Complex