Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme
Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme
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DOI：
10.1016/j.pep.2008.11.010
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发表时间：
2009-06-01
影响因子：
1.6
通讯作者：
da Silva, Aline M.
中科院分区：
文献类型：
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作者：
de Almeida, Fabiana M.;Bonini, Beatriz M.;da Silva, Aline M.
来自粗糙脉孢菌的中性海藻糖酶在大肠杆菌中表达为类似于84kDa的多肽，与根据相应cDNA计算的理论大小一致。通过亲和层析纯化的重组中性海藻糖酶表现出80-150 mU/mg蛋白质的比活性。最适pH和温度分别为7.0和30℃。该酶对海藻糖具有绝对特异性，对40℃培养相当敏感。重组酶完全依赖于钙，并受到ATP、铜、银、铝和钴的抑制。 K-M 为 42 mM，V-max 为 30.6 nmol 葡萄糖/分钟。重组蛋白被cAMP依赖性蛋白激酶磷酸化，但没有显着激活。使用针对重组蛋白制备的多克隆抗血清进行的免疫印迹表明，中性海藻糖酶蛋白水平在粗糙脉孢菌生长的指数期增加，并在稳定期下降。这是在大肠杆菌中产生的中性海藻糖酶的首次报道，其具有与真菌天然中性海藻糖酶相似的生化特性，包括钙依赖性。 (C) 2008 Elsevier Inc. 保留所有权利。
Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of similar to 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K-M was 42 mM, and V-max was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence. (C) 2008 Elsevier Inc. All rights reserved.