腾冲嗜热厌氧菌耐热解旋酶Tte-uvrD的克隆表达及粗酶的初步应用  

Clone of Tte-uvrD from Thermoanaerobacter tengcongensis and activity of crude enzyme

作　　者：章丽[1] 余以刚[1] 黄秀丽[2] 肖性龙[1] 

机构地区：[1]华南理工大学轻工与食品学院,广东广州510640 [2]广东省惠州市质量计量监督检测所,广东惠州516000

出　　处：《食品工业科技》2014年第12期226-229,235,共5页Science and Technology of Food Industry

基　　金：国家自然科学基金青年科学基金资助项目(31101279);教育部高等学校博士学科点专项科研基金新教师类资助课题(20110172120034);广东省科技计划资助项目(2011B020314004);华南理工大学中央高校基本科研业务费重点项目(2013ZZ068);惠州市科技计划项目(0031939140712048)

摘　　要：以腾冲嗜热厌氧菌(Thermoanaerobacter tengcongensis)基因组DNA为模版,通过PCR克隆编码解旋酶Tte-uvrD的基因tte-uvrd,将该基因插入原核表达载体pET-32a(+),构建重组表达载体pET-32a(+)-tte-uvrd,转入E.coli BL21中,经蓝白斑筛选和双酶切法筛选阳性重组转化子,挑取测序正确的单个阳性菌落,在IPTG诱导下表达出重组蛋白Tte-uvrD。诱导后的菌体经超声破碎和离心,上清液用硫酸铵沉淀法初步纯化,透析除盐,冻干复溶后得到粗酶液。用tHDA反应来验证Tte-uvrD粗酶液的活性,比较不同储存温度下粗酶液酶活保持的时间,并比较-20℃下储存两个月的粗酶液与商品化纯酶的tHDA反应灵敏度。结果表明,用本研究建立的克隆方法可成功克隆出耐热解旋酶TteuvrD,其粗酶液能用于tHDA反应,说明粗酶液具有解旋活性;粗酶液在-20℃下第80d酶活仍能保持稳定,4℃下则只能保持酶活约一周;-20℃下储存两个月的粗酶液能达到与商品化纯酶相当的灵敏度。In order to obtain the crude Tte-uvrD helicase,gene tte-uvrd was amplified from Thermoanaerobacter tengcongensis by PCR,and was cloned into prokaryotic expression vector pET-32a（＋）. Apart from screening by the X-Gal method and double restriction enzyme digestion method ,a two-direction sequencing was performed. The recombined vectors pET-32a（＋）-tte-uvrd was transformed to E.Cofi BL21 strains and were induced by IPTG. The crude enzyme of Tte-uvrD was obtained by ultrasonication,centrifugation,sedimentation of saturation ammonium sulfate,dialysis and freeze-drying. The tHDA reactions were conducted to verify the activity of crude enzyme,as well as to compare the activity between crude enzyme and commercial pure enzyme. Activities of crude enzyme in different storage temperetures were also compared. The results showed that tte-uvrd gene was successfully cloned and Tte-uvrD helicase was expressed. The crude enzyme displayed a good activity in the tHDA assay,indicating that the helicase had the unwinding activity and the activity stays stable for about 80d in -20℃,but only about one week in 4℃. The crude enzyme displayed a similar sensitivity as the commercial pure enzyme after stored in -20℃ for 80d.

关 键 词：腾冲嗜热厌氧菌 Tte-uvrD 克隆表达 粗酶 tHDA 

分 类 号：TS207.4[轻工技术与工程—食品科学]