Characterization of a cytoplasmic trehalase of Escherichia coli
Characterization of a cytoplasmic trehalase of Escherichia coli
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DOI：
10.1128/jb.178.21.6250-6257.1996
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发表时间：
1996-11-01
影响因子：
3.2
通讯作者：
Boos, W
中科院分区：
文献类型：
--
作者：
Horlacher, R;Uhland, K;Boos, W
大肠杆菌能在渗透胁迫下合成海藻糖，并能利用海藻糖作为碳源，在低渗透和高渗透压下利用海藻糖的途径不同，在高渗透压下，诱导产生周质海藻糖酶(TreA)，将周质中的海藻糖分解为葡萄糖，葡萄糖被磷酸转移酶系统吸收，在低渗透压下，海藻糖被磷酸转移酶系统中的海藻糖专一性酶II吸收为海藻糖-6-磷酸，然后被水解为葡萄糖和葡萄糖-6-磷酸。我们报道了一种新的胞质海藻糖水解酶，该酶编码基因tref是在NLa启动子控制下被克隆的，该酶是从一株高表达菌株的提取液中纯化出来的，并对其生化特性进行了研究。该酶对海藻糖的K-m值为1.9 mM，每毫克蛋白质的V-max为54mU/min，酶为单体，最适pH为6.0，不依赖于金属；Tref的相对分子质量为63,703(549个氨基酸)，与Trea高度同源；Tref的不同氨基酸比Trea具有更强的极性和酸性；分离出了产生比野生型多17倍的Tref的突变体。
Escherichia coli can synthesize trehalose in response to osmotic stress and is able to utilize trehalose as a carbon source, The pathway of trehalose utilization is different at low and high osmolarity, At high osmolarity, a periplasmic trehalase (TreA) is induced that hydrolyzes trehalose in the periplasm to glucose, Glucose is then taken up by the phosphotransferase system, At low osmolarity, trehalose is taken up by a trehalose-specific enzyme II of the phosphotransferase system as trehalose-6-phosphate and then is hydrolyzed to glucose and glucose-6-phosphate. Here we report a novel cytoplasmic trehalase that hydrolyzes trehalose to glucose, treF, the gene encoding this enzyme, was cloned under nl a promoter control, The enzyme (TreF) was purified from extracts of an overexpressing strain and characterized biochemically. It is specific for trehalose exhibiting a K-m of 1.9 mM and a V-max of 54 mu mol of trehalose hydrolyzed per min per mg of protein, The enzyme is monomeric, exhibits a broad pH optimum at 6.0, and shows no metal dependency, TreF has a molecular weight of 63,703 (549 amino acids) and is highly homologous to TreA, The nonidentical amino acids of TreF are more polar and more acidic than those of TreA, The expression of treF as studied by the expression of a chromosomal treF-lacZ fusion is weakly induced hy high osmolarity of the medium and is partially dependent on RpoS, the stationary-phase sigma factor, Mutants producing 17-fold more TreF than does the wild type were isolated.