假单胞菌生物合成L-半胱氨酸途径中脱巯基酶的研究  

Research of Desulfhydrase Involved in L-cysteine Biosynthetic Pathway in Pseudomonas sp.

作　　者：余养盛[1] 李洋[1] 金永杰[1] 白钢[1] 杨文博[1] 

机构地区：[1]南开大学生命科学学院,天津300071

出　　处：《微生物学通报》2006年第4期21-26,共6页Microbiology China

基　　金：国家自然科学基金资助项目(No.30470053);天津市重点基金资助项目(No.05YFJZJC00900)

摘　　要：通过PCR方法扩增得到假单胞菌TS1138L-半胱氨酸脱巯基酶基因(cd),将其克隆至pBluescript SKII载体,测定了含有L-半胱氨酸脱巯基酶基因的1·2kb DNA片段序列,并与其它菌株的脱巯基酶基因进行了同源性比较;同时,将其克隆至表达载体pET-21a(+),IPTG诱导表达,表达产物经Ni-NTA柱亲合层析后,得到纯化的重组蛋白。利用脱巯基酶的活性染色方法对重组表达的L-半胱氨酸脱巯基酶进行了鉴定,并探讨了L-半胱氨酸脱巯基酶的酶学性质,以及在生物转化合成L-半胱氨酸途径中的关键作用。The L-cysteine desulfhydrase gene （cd） of Pseudomonas sp. TS1138 was amplified by PCR, and the amplified gene was recombined in the cloning vector pBluescript SKIL The 1.2kb DNA fragment containing cd was sequenced, and its homology with other dasulfhydrases was blast; then the cd was cloned into the expression vector pET-21a （ ＋ ）, and afterward expressed by IPTG inducement. The expression protein was purified by Ni- NTA His-Bind Resin. Then the expression protein was identified by the method of activity staining of desulfhydrase, and the characterization of L-cysteine desulfhydrase and the critical role it played in the L-cysteine biosynthetic pathway were discussed.

关 键 词：L-半胱氨酸脱巯基酶 假单胞菌 基因克隆 基因表达 酶学性质 

分 类 号：Q936[生物学—微生物学]