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1、眉題2通過同源介導(dǎo)a-淀粉酶基因擴增改良地衣芽孢桿菌a-淀粉酶生產(chǎn)菌株牛丹丹,石貴陽,王正祥江南大學(xué)生物工程學(xué)院生物資源與生物能源研究中心和工業(yè)生物技術(shù)教育部重點實驗室,無錫214122摘要:地衣芽孢桿菌高溫a-淀粉酶(BLA)是淀粉水解與生物加工過程中重要關(guān)鍵酶制劑之一。為了進一步提高地衣芽孢桿菌高溫a-淀粉酶生產(chǎn)菌株的生產(chǎn)性能,本研究構(gòu)建了一種含有地衣芽孢桿菌高溫a-淀粉酶編碼基因amyL的整合性重組質(zhì)粒pBL-amyL。將重組質(zhì)粒pBL-amyL轉(zhuǎn)化入BLA工業(yè)生產(chǎn)菌株BacilluslicheniformisB0204,再在卡那霉
2、素存在下介導(dǎo)其B.licheniformisB0204染色體中的同源整合與高溫a-淀粉酶編碼基因amyL的擴增,由此獲得了攜帶多個amyL拷貝的轉(zhuǎn)化子。對轉(zhuǎn)化子的amyL拷貝數(shù)及其BLA發(fā)酵水平分別用熒光實時定量PCR及搖瓶發(fā)酵試驗進行評價與鑒定。與出發(fā)菌株B0204相比,含2~5倍amyL拷貝數(shù)的重組菌的BLA的合成水平顯著提高。其中,重組菌REBL18生產(chǎn)BLA的水平提高了89.2%。關(guān)鍵詞:地衣芽孢桿菌a-淀粉酶,基因擴增,發(fā)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:通過同源介導(dǎo)a-淀粉酶基因擴增改良地衣芽孢桿菌a-淀粉酶生產(chǎn)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:通過同源介導(dǎo)a-淀粉酶基因擴增改良地衣芽孢桿菌a-淀粉酶生產(chǎn)菌株379microorganisms,plantsandanimals.a-amylaseandtherelatedamylolyticenzymesarewidelyusedinbiotechnologyforstarchdegradation[1]andinsyntheticchemistr
8、yfortheformationofoligosaccharidesbytransglycosylation[2].Oneofmajorapplicationsoftheenzymeistol
