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  

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

作　　者：马挺[1] 王仁静[1] 李京浩[1] 梁凤来[1] 张心平[1] 刘如林[1] 

机构地区：[1]南开大学生命科学学院,天津300071

出　　处：《南开大学学报（自然科学版）》2005年第6期1-6,共6页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：国家自然科学基金资助项目(20374032);天津市自然科学基金资助项目(05YFJMJC00700)

摘　　要：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.DszC catalyzing, the first, also the key step in the microbial DBT desulfurization is the conversion of DBT to DBT sulfone （DBTO2）. 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 methyl-DBT, but not DBT derivates such as DBF which is no sulfur and inorganic sulfur.

关 键 词：生物脱硫 红球菌 二苯并噻吩 单加氧酶 

分 类 号：Q93[生物学—微生物学]