Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa
Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa
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荧光假单胞菌亚种中多个羧甲基纤维素酶基因的证据。
DOI：
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发表时间：
1987
期刊：
影响因子：
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通讯作者：
Judith Hall
中科院分区：
文献类型：
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作者：
H. Gilbert;G. Jenkins;D. Sullivan;Judith Hall
荧光假单胞菌亚种基因组文库。在噬菌体λ47.1中构建了cellulosa DNA，并分离出表达羧甲基纤维素酶（CMCase）活性的重组体。将3个噬菌体中7.3 kb的EcoRI片段、9.4 kb的EcoRI片段和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CMCase基因在三个质粒中的位置。pjh1包含两个不同的DNA区域编码cmcase，它们由相同的启动子控制。克隆的4种酶均能裂解对硝基苯-β- d -葡萄糖苷，虽然裂解速率很低，但均未表现出外葡聚糖酶活性。与在大肠杆菌中克隆的其他细胞外酶一样，所有cmcase都被输出到肠道细菌的质周空间。由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抑制。
SummaryA genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in bacteriophage λ47.1 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-β-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.