海栖热袍菌磷酸戊糖变位酶基因克隆表达及发酵条件优化  

Cloning,Expression and Fermentation Optimization of Thermotoga Maritima Phosphpentomutase Gene in E. coli

作　　者：梅旭旭 陈婷[2] 孙芳芳[1] 张兴群[1,2] MEI Xu-xu CHEN Ting SUN Fang-fang ZHANG Xing-qun(Key Laboratory of Science ＆ Technology of Eco-Textile （Donghua University/Jiangnan University） of Ministry of Education, Shanghai 201620, China College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China)

机构地区：[1]生态纺织教育部重点实验室(东华大学/江南大学),上海201620 [2]东华大学化学化工与生物工程学院,上海201620

出　　处：《食品与药品》2017年第4期229-234,共6页Food and Drug

基　　金：国家科技重大专项"重大新药创制"项目资助(2014ZX09J14103-09C-003)

摘　　要：目的从嗜热微生物海栖热袍菌中克隆磷酸戊糖变位酶基因,实现其在大肠杆菌中的表达。方法以海栖热袍菌基因组DNA为模板,利用PCR技术扩增获得编码磷酸戊糖变位酶蛋白基因(ppm),连接到具有T7强启动子的p ET-32a质粒中,构建重组工程菌E.coli BL 21(DE3){p ET-32a-Tmppm}。通过Plackett-Burman设计、最陡爬坡试验和Box-Behnken试验方法优化工程菌发酵条件,构建回归方程。结果双酶切鉴定、测序分析和十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)证实海栖热袍菌磷酸戊糖变位酶基因可在工程菌中表达。最佳发酵条件为诱导种龄0.6,诱导温度22.5℃,诱导时间13 h,异丙基β-D-硫代半乳糖苷(IPTG)浓度0.45 mmol/L,培养基初始p H 7.6,接种量2.25%。结论发酵条件优化后磷酸戊糖变位酶表达量大幅提高。实验结果为该酶的性质研究和应用提供了支撑。Objective To achieve the expression of Thermotoga maritima phosphpentomutase gene in engineered E. coli. Methods The gene encoding the phosphopentomutase （ppm） was obtained by PCR amplification using genomic DNA of Thermotoga maritima as template; then ppm was connected to plasmid pET-32a to construct recombinant engineering strain E. coli BL21 （DE3）{pET-32a-Tmppm}. Plackett-Burrnan （PB） design, the steepest ascent path and Box-Behnken tests were used to optimize the liquid submerged fermentation conditions. Results The expression of the phosphpentomutase gene in the recombinant engineering strain was proved by double enzyme digestion identification, sequencing and SDS-PAGE.The optimum fermentation parameters were as follows： induction age 0.6, induction temperature 22.5 ℃, initial pH 7.6, inoculation amount 2.25 % and induction time 13 h with 0.45 mmol/L IPTG. Conclusion The yield of phosphpentomutase is greatly improved under optimum fermentation condition. This study provides a basic work for the production of Thermotoga maritima phosphpentomutase by engineered E. coli strain inindustrial scale.

关 键 词：海栖热袍菌 磷酸戊糖变位酶 重组大肠杆菌 发酵优化 

分 类 号：Q558[生物学—生物化学]