Alkaline phosphatase from the Antarctic strain TAB5 - Properties and psychrophilic adaptations
Alkaline phosphatase from the Antarctic strain TAB5 - Properties and psychrophilic adaptations
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DOI：
10.1046/j.1432-1327.2000.01127.x
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发表时间：
2000-02-01
期刊：
影响因子：
--
通讯作者：
Bouriotis, V
中科院分区：
文献类型：
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作者：
Rina, M;Pozidis, C;Bouriotis, V
从嗜冷菌TABS中克隆了碱性磷酸酶（AP）基因，并测定了其核苷酸序列。结果表明，该基因有一个1125个碱基的开放阅读框，编码一个由22个氨基酸组成的信号肽和一个353个氨基酸组成的成熟蛋白。推导的AP蛋白序列与枯草芽孢杆菌的AP III和AP IV序列具有38%的同一性，并保留了来自各种来源的AP的核心结构和活性位点的典型序列基序。基于突变大肠杆菌AP D153 H的晶体结构，构建了TABS AP的三维同源模型，并在此基础上对该酶的氨基酸序列进行了分析，以解释其可能的嗜冷适应性。coliBL21（DE3）细胞，从细胞膜组分中分离重组蛋白，并对其性质进行了检测。纯化的TABS AP显示出冷酶的典型特征：在低温下具有高催化活性和显著的热敏性。
The gene encoding alkaline phosphatase (AP) from the psychrophilic strain TABS was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1125 base pairs which encodes a polypeptide consisting of signal peptide of 22 amino acids and a mature protein of 353 amino acids was identified. The deduced protein sequence of AP exhibits a 38% identity to the AP III and AP IV sequences of Bacillus subtilis and conserves the typical sequence motifs of the core structure and active sites of APs from various sources. Based on the crystal structure of the mutated Escherichia coli AP D153H, a homology-based 3D model of the TABS AP was constructed on the basis of which various features of the enzyme amino-acid sequence can be interpreted in terms of potential psychrophilic adaptations.The AP gene was expressed in E. coli BL21(DE3) cells, the recombinant protein was isolated to homogeneity from the membrane fraction of the cells and its properties were examined. The purified TABS AP shows typical features of a cold enzyme: high catalytic activity at low temperature and a remarkable thermosensitivity.The use of this heat-labile enzyme, for dephosphorylation of nucleic acids, simplifies dephosphorylation protocols.