二苯并噻吩单加氧酶基因(dszC)的克隆及其在大肠杆菌中的表达

利用PCR法从红球菌DS-3中克隆了dszC基因,序列测定结果与报道的Rhod ococcus ery throp olisIGTS8中d szC基因有99%的同源性.构建了含dszC基因的表达载体paC 5并在E.coli中实现高效表达,重组蛋白(D szC)在降温条件下重组菌表达量可达20%.可溶性D szC经N i2 亲和层析纯化达到电泳纯(95%以上).用纯化的D szC制备一抗血清进行免疫印记检测,证明工程菌表达产物中存在目的蛋白.纯化的D szC酶活可达0.36 U.D szC能作用于DBT及其甲基取代物,但不能利用无硫的DBT类似物及无机硫源.

Cloning of DBT Monooxygenase Gene (dszC) from Rhodococcus sp. DS-3 and Its Expression in E. coli

DszC catalyzing,the first,also the key step in the microbial DBT desulfurization is the conversion of DBT to DBT sulfone(DBTO_2).In this study,dszC of a DBT-desulfurizing bacterium Rhodococcus sp.DS-3 was cloned by PCR.The sequence cloned was 99 % homologue to Rhodococcus erythrolis IGTS8 that reported in Genebank.the gene dszC could be overexpressed effectively after inserted into plasmid pET28a( ) and transformed into E.coli BL21 strain.The expression amount of DszC was about 20 % of total supernatant at low temperature.The soluble DszC in the supernatant was purified by Ni~(2 ) chelating His-Tag Resin column and SDS-PAGE to electronics pure.Only one band was detected by western-blotting which antibody raised in mouse against purified DszC in expression product of BL21(DE3,paC5) and Rhodococcus sp. DS-3.The activity of purified DszC was 0.36 U.The DszC can utilize the organic compound such as DBT and methy1-DBT,but not DBT derivates such as DBF which is no sulfur and inorganic sulfur.