通过同源介导-淀粉酶基因扩增改良地衣芽孢杆菌.DOCVIP

通过同源介导?-淀粉酶基因扩增改良地衣芽孢杆菌?-淀粉酶生产菌株 牛丹丹, 石贵阳, 王正祥 江南大学生物工程学院 生物资源与生物能源研究中心和工业生物技术教育部重点实验室, 无锡 214122 摘 要: 地衣芽孢杆菌高温?-淀粉酶(BLA)是淀粉水解与生物加工过程中重要关键酶制剂之一。为了进一步提高地衣芽孢杆菌高温?-淀粉酶生产菌株的生产性能, 本研究构建了一种含有地衣芽孢杆菌高温?-淀粉酶编码基因amyL的整合性重组质粒pBL-amyL。将重组质粒pBL-amyL转化入BLA工业生产菌株Bacillus licheniformis B0204, 再在卡那霉素存在下介导其B. licheniformis B0204染色体中的同源整合与高温?-淀粉酶编码基因amyL的扩增, 由此获得了携带多个amyL拷贝的转化子。对转化子的amyL拷贝数及其BLA发酵水平分别用荧光实时定量PCR及摇瓶发酵试验进行评价与鉴定。与出发菌株B0204相比, 含2~5倍amyL拷贝数的重组菌的BLA的合成水平显著提高。其中, 重组菌REBL18生产BLA的水平提高了89.2%。 关键词: 地衣芽孢杆菌?-淀粉酶, 基因扩增, 发酵 Genetic improvement of α-amylase producing Bacilluslicheniformis by homolog-mediated α-amylase geneamplification Dandan Niu, Guiyang Shi, and Zhengxiang Wang Center for Bioresource and Bioenergy, School of Biotechnology and the Key Laboratory of Industrial Biotechnology of the Ministry of Education, Jiangnan University, Wuxi 214122, China Abstract: Bacillus licheniformis ?-amylase (BLA) is one of the most important enzymes involved in starch hydrolysis and many biotechnological processes. To improve the BLA productivity, an integrative plasmid pBL-amyL carrying amyL gene encoding a thermophilic ?-amylase of B. licheniformis was constructed and transformed into B. licheniformis B0204, an industrial α-amylase producer. The transformants harboring different copies of amyL were developed on kanamycin by using homolog-mediated chromosomal amplification of ?-amylase gene. The recombinants with different multiple copies of amyL integrated in the chromosome were identified by real-time PCR and evaluated by shake-flask fermentation. Recombinants harboring 2?5 multiple copies of amyL produced more ?-amylase comparison to the parental strain B0204. Keywords: Bacillus licheniformis ?-amylase, gene amplification, fermentation ?-amylase (EC ) is an endo-1,4-?-D-gluco- hydrolytic enzyme, which occurs widely in microorganisms, plants and animals. ?-amylase and the related amylolytic enzymes are widely used in biote