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

钝齿棒杆菌（Corynebacterium crenatum）SYPA是本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，本文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1434 bp，编码476个氨基酸，理论蛋白分子量大小为50.8 kDa，其与C. glutamicum ATCC 13032比对其同源性为99.4%，相差10 bp，3个氨基酸。将其在E.coli BL21(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40.9 g/L，比出发菌株产量的35.8 g/L提高了约14.2%。

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

 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%.