Polysaccharide lyase: molecular cloning of gellan lyase gene and formation of the lyase from a huge precursor protein in Bacillus sp. GL1.
Polysaccharide lyase: molecular cloning of gellan lyase gene and formation of the lyase from a huge precursor protein in Bacillus sp. GL1.
复制标题
多糖裂解酶：结冷胶裂解酶基因的分子克隆以及芽孢杆菌中巨大前体蛋白的裂解酶的形成。
DOI：
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发表时间：
1998
影响因子：
3.9
通讯作者：
K. Murata
中科院分区：
文献类型：
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作者：
W. Hashimoto;N. Sato;S. Kimura;K. Murata
一种细菌，芽孢杆菌属GL 1，组成型产生分子量为140 kDa的胞外多糖降解酶（结冷胶裂解酶）。使用粘粒载体Charomid 9-36在大肠杆菌中构建细菌的基因组DNA文库。通过在大肠杆菌中筛选结冷胶降解表型，克隆了编码裂合酶的基因。coli细胞，测定了该基因的核苷酸序列。该基因含有一个开放阅读框，由7425个碱基组成，编码一个分子量为263 kDa的多肽。该多肽含有与酶的N-末端氨基酸序列相同的氨基酸序列，并且与任何先前公布的蛋白质序列没有同源性。E.用该基因转化的大肠杆菌细胞表现出结冷胶裂解酶活性，并在细胞内产生分子量约为260 kDa的蛋白质。纯化的蛋白质，并显示出具有密切相似的酶的性质，从芽孢杆菌属GL 1的天然酶的最佳pH值和温度的活动，底物特异性，和酶的作用模式。这些结果表明，在芽孢杆菌GL 1中，结冷胶裂解酶首先作为巨大的前体蛋白（263 kDa）产生，然后通过切除约120 kDa的C-末端肽将蛋白质后处理成胞外成熟形式（140 kDa）。
A bacterium, Bacillus sp. GL1, produced constitutively the extracellular polysaccharide-degrading enzyme (gellan lyase) with a molecular mass of 140 kDa. A genomic DNA library of the bacterium was constructed in Escherichia coli using the cosmid vector, Charomid 9-36. The gene encoding the lyase was cloned by screening for a gellan-degrading phenotype in E. coli cells and the nucleotide sequence of the gene was determined. The gene contained an open reading frame consisting of 7425 base pairs coding a polypeptide with a molecular mass of 263 kDa. The polypeptide contained the same amino acid sequence as N-terminal amino acid sequence of the enzyme and exhibited no homology with any previously published protein sequences. E. coli cells transformed with the gene exhibited gellan lyase activity and produced a protein with a molecular mass of about 260 kDa intracellularly. The protein was purified and shown to have the closely similar enzymatic properties to those of the native enzyme from Bacillus sp. GL1 with respect to optimal pH and temperature for activity, substrate specificity, and the mode of enzyme action. These results suggest that, in Bacillus sp. GL1, gellan lyase is first produced as a huge precursor protein (263 kDa) and then the protein is posttranslationally processed into extracellular mature form (140 kDa) through excising C-terminal peptide of about 120 kDa.