Petersen D J, Bennett G N
Department of Biochemistry, Rice University, Houston, Texas 77251.
Appl Environ Microbiol. 1990 Nov;56(11):3491-8. doi: 10.1128/aem.56.11.3491-3498.1990.
In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment. A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.
在丙酮丁醇梭菌ATCC 824中，乙酰乙酸脱羧酶（EC 4.1.1.4）对于溶剂生产至关重要，它催化乙酰乙酸脱羧生成丙酮。我们在此报告了从丙酮丁醇梭菌ATCC 824中纯化该酶以及在大肠杆菌中克隆和表达编码乙酰乙酸脱羧酶的基因。使用从纯化蛋白获得的N端氨基酸序列衍生的寡脱氧核苷酸探针，通过噬菌斑杂交筛选丙酮丁醇梭菌DNA的噬菌体λEMBL3文库。通过Southern杂交分析来自阳性噬菌斑的噬菌体DNA。对与探针杂交的DNA片段进行限制性图谱分析和随后的亚克隆，将该基因定位在一个约2.1 kb的EcoRI/Bg/II片段内。在含有梭菌基因的大肠杆菌全细胞提取物的Western印迹（免疫印迹）和大细胞分析中均观察到一种分子量约为28,000的多肽，与纯化的乙酰乙酸脱羧酶相对应。尽管该基因在丙酮丁醇梭菌中表达受到严格调控，但它在大肠杆菌中表达良好，尽管其启动子序列源自梭菌。
