Gilbert H J, Jenkins G, Sullivan D A, Hall J
Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, UK.
Mol Gen Genet. 1987 Dec;210(3):551-6. doi: 10.1007/BF00327211.
A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in bacteriophage lambda 47.1 and recombinants expressing carboxymethylcellulase (CMCase) activity isolated. A 7.3 kb partial EcoRI fragment, a 9.4 kb EcoRI fragment and a 5.8 kb HindIII fragment were subcloned from three different phages into pUC18 to yield recombinant plasmids pJHH1, pJHH3 and pGJH2 respectively. Cells of Escherichia coli harbouring these plasmids expressed CMCase activity. The positions of the CMCase genes in the three plasmids were determined by subcloning and transposon mutagenesis. pJHH1 contained two distinct DNA regions encoding CMCases, which were controlled by the same promoter. All four cloned enzymes cleaved p-nitrophenyl-beta-D-glucopyranoside, although at a very low rate, but none exhibited exoglucanase activity. In common with other extracellular enzymes cloned in E. coli, all the CMCases were exported to the periplasmic space in the enteric bacterium. The carboxymethylcellulase genes encoded by pJHH1 and pJHH3, were subject to glucose repression in E. coli.
构建了荧光假单胞菌纤维素亚种DNA的基因组文库，该文库采用噬菌体λ47.1构建，并分离出表达羧甲基纤维素酶（CMCase）活性的重组体。从三个不同的噬菌体中分别将一个7.3 kb的EcoRI部分片段、一个9.4 kb的EcoRI片段和一个5.8 kb的HindIII片段亚克隆到pUC18中，分别得到重组质粒pJHH1、pJHH3和pGJH2。携带这些质粒的大肠杆菌细胞表达CMCase活性。通过亚克隆和转座子诱变确定了三个质粒中CMCase基因的位置。pJHH1包含两个不同的编码CMCase的DNA区域，它们由同一个启动子控制。所有四种克隆酶都能切割对硝基苯基-β-D-吡喃葡萄糖苷，尽管切割速率非常低，但均未表现出外切葡聚糖酶活性。与在大肠杆菌中克隆的其他细胞外酶一样，所有CMCase都被转运到肠道细菌的周质空间。pJHH1和pJHH3编码的羧甲基纤维素酶基因在大肠杆菌中受到葡萄糖阻遏。
