PECTATE LYASE FROM BACILLUS-SUBTILIS - MOLECULAR CHARACTERIZATION OF THE GENE, AND PROPERTIES OF THE CLONED ENZYME
PECTATE LYASE FROM BACILLUS-SUBTILIS - MOLECULAR CHARACTERIZATION OF THE GENE, AND PROPERTIES OF THE CLONED ENZYME
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DOI：
10.1016/0014-5793(93)80410-v
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发表时间：
1993-12-13
期刊：
影响因子：
3.5
通讯作者：
ROBERTBAUDOUY, J
中科院分区：
文献类型：
--
作者：
NASSER, W;AWADE, AC;ROBERTBAUDOUY, J
果胶酸裂解酶（PL）通过裂解植物细胞壁的主要成分果胶而引发植物软腐病。本文首次从芽孢杆菌属中克隆了果胶酸裂解酶基因。该基因是从构建在pUC 18载体上的枯草芽孢杆菌基因组文库中分离得到的。通过Southern杂交显示该基因在B中以单拷贝存在。枯草杆菌基因组一个1.6kb对的HindIII限制性片段的核苷酸序列，该片段赋予E.大肠杆菌中，显示了1，260 bp的开放阅读框，编码420个氨基酸的多肽，该多肽包括21个氨基酸的信号序列。推导的45，605 Da蛋白显示与来自欧文氏菌的PL的同源性。B。subtilis PL克隆到E.大肠杆菌位于周质中。从E.用pT7系统过量生产后的大肠杆菌周质液。纯化的酶的生化性质与从B分离的PL中发现的那些相似。枯草杆菌胞外培养基
Pectate lyases (PL) initiate soft-rot diseases in plants by cleaving pectin which is the major component of the plant cell wall. The present paper reports the first cloning and characterization of a pectate lyase (pel) gene from the Bacillus genus. This gene was isolated from a Bacillus subtilis genomic library constructed in pUC18 as Vector and Escherichia coli as host. By Southern hybridization this gene was shown to be present in a single copy in the B. subtilis genome. The nucleotide sequence of a 1.6 kb-pair HindIII restriction fragment, which confers pectate lyase activity to E. coli, indicated a 1,260 bp open reading frame encoding a 420 amino acid polypeptide which includes a 21 amino acid signal sequence. The 45,605 Da deduced protein displays homologies to PLs from Erwinia chrysanthemi. The B. subtilis PL cloned in E. coli was located in the periplasm. It was purified to homogeneity in a one-step procedure from the E. coli periplasmic fluid after overproduction using the pT7 system. Biochemical properties of the purified enzyme were similar to those found for the PL isolated from B. subtilis extracellular media.