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1、眉题2通过同源介导a-淀粉酶基因扩增改良地衣芽孢杆菌a-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a-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a-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a-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BacilluslicheniformisB0204,再在卡那霉
2、素存在下介导其B.licheniformisB0204染色体中的同源整合与高温a-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a-淀粉酶,基因扩增,发酵Geneticimprovementofα-amylaseproducingBacilluslicheni
3、formisbyhomolog-mediatedα-amylasegeneamplificationDandanNiu,GuiyangShi,andZhengxiangWangCenterforBioresourceandBioenergy,SchoolofBiotechnologyandtheKeyLaboratoryofIndustrialBiotechnologyoftheMinistryofEducation,JiangnanUniversity,Wuxi214122,ChinaAbstract:Bacilluslichenifo
4、rmisa-amylase(BLA)isoneofthemostimportantenzymesinvolvedinstarchhydrolysisandmanybiotechnologicalprocesses.ToimprovetheBLAproductivity,anintegrativeplasmidpBL-amyLcarryingamyLgeneencodingathermophilica-amylaseofB.licheniformiswasconstructedandtransformedintoB.licheniformi
5、sB0204,anindustrialα-amylaseproducer.ThetransformantsharboringdifferentcopiesofamyLweredevelopedonkanamycinbyusinghomolog-mediatedchromosomalamplificationofa-amylasegene.TherecombinantswithdifferentmultiplecopiesofamyLintegratedinthechromosomewereidentifiedbyreal-timePCRa
6、ndevaluatedbyshake-flaskfermentation.Recombinantsharboring2−5multiplecopiesofamyLproducedmorea-amylasecomparisontotheparentalstrainB0204.Keywords:Bacilluslicheniformisa-amylase,geneamplification,fermentationJournals.im.ac.cn牛丹丹等:通过同源介导a-淀粉酶基因扩增改良地衣芽孢杆菌a-淀粉酶生产菌株379a-amylas
7、e(EC3.2.1.1)isanendo-1,4-a-D-gluco-hydrolyticenzyme,whichoccurswidelyinJournals.im.ac.cn牛丹丹等:通过同源介导a-淀粉酶基因扩增改良地衣芽孢杆菌a-淀粉酶生产菌株379microorganisms,plantsandanimals.a-amylaseandtherelatedamylolyticenzymesarewidelyusedinbiotechnologyforstarchdegradation[1]andinsyntheticchemistr
8、yfortheformationofoligosaccharidesbytransglycosylation[2].Oneofmajorapplicationsoftheenzymeistol
