钝齿棒杆菌精氨酸琥珀酸酶编码基因argH的克隆表达及其重组菌发酵产精氨酸研究  

Cloning,Expression and Analysis of the argH Gene Encoding Argininosuccinate Lyase from Corynebacterium crenatum

作　　者：饶志明[1] 徐美娟[1] 陆元修[1] 周晨[1] 蓝春燕[1] 窦文芳 张晓梅 许泓瑜 许正宏[1,2] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,无锡214122 [2]江南大学医药学院制药工程实验室,无锡214122

出　　处：《中国生物工程杂志》2010年第9期49-55,共7页China Biotechnology

基　　金：国家自然科学基金(20676053;30970056);国家"863"计划(2006AA020103;2007AA02Z207);国家"973"计划(2007CB707804);霍英东青年基金(121020)资助项目

摘　　要：钝齿棒杆菌(Corynebacterium crenatum)SYPA是实验室筛选获得的一株高产精氨酸生产菌株。精氨酸琥珀酸酶(AL)是精氨酸合成过程中的最后一个酶,催化底物精氨酸琥珀酸生成产物精氨酸。为进一步提高精氨酸产量,以钝齿棒杆菌基因组为模板,扩增得到其编码基因argH,全长为1434bp,编码476个氨基酸,理论蛋白分子量大小为50.8kDa,其与C.glutamicum ATCC13032比对其同源性为99.4%,相差10bp,3个氨基酸。将其在E.coliBL21(DE3)及C.crenatum SYPA中成功表达。利用载体pET-28a上的6×His·Tag选用Ni柱亲和层析纯化AL,纯化后获得的重组蛋白的比酶活达156.9mU/mg蛋白,总回收率为72.3%,对该酶的部分酶学性质进行了初步研究,并发现产物精氨酸对其具有反馈抑制作用。成功构建钝齿棒杆菌重组穿梭表达质粒pJC1-tac-argH并将其通过电击转化法转入C.crenatum SYPA中,加强其代谢途径中AL蛋白表达量,并对其发酵产精氨酸做了初步分析。结果表明与出发菌株相比,转化子在精氨酸琥珀酸酶酶活增强了66.8%的基础上精氨酸产量达40.9g/L,比出发菌株产量的35.8g/L提高了约14.2%。Argininosuccinate lyase （EC 4.3.2.1;AL） genes from L-arginine producing mutant Corynebacterium crenatum SYPA were cloned and sequenced.Analysis of argH sequences revealed that only one ORF existed,which used ATG as the initiation codon and coded a peptide of 476 amino acids with acalculated molecular weight of 50.8 kDa.Only 10 nucleotide difference was found in the structure gene and the difference caused 3 change of amino acid by comparision of the gene sequences between C.crenatum SYPA and the Corynebacterium glutamicum ATCC 13032.The ORF sequence of argHs showed homologies of 99.4%.The argH gene from C.crenatum was expressed both in E.coli and C.crenatum SYPA.Then AL was purified by Ni-NTA affinity chromatography and the enzymatic characterization of it was determines.The expression vector pJC1-tac-argH was transducted to C.crenatum SYPA.The AL was expressed well and the activity was improved by 66.8%.The fermentive character of CCH1 （pJC1-tac-argH） was also primary analysed.The result shows that the acid producing ability of recombinant strain is improved by 14.2%.

关 键 词：L-精氨酸 钝齿棒杆菌 精氨酸琥珀酸酶 酶学性质 发酵 

分 类 号：Q78[生物学—分子生物学]