Clone, Purification and Characterization of Thermostable Aminopeptidase ST1737 from Sulfolobus tokodaii  

作　　者：YU Xiaoxiao HAO Weiwei XIE Guiqiu WANG Yingwu GAO Renjun 

机构地区：[1]Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130012, P. R. China [2]College of Pharmacy, Jilin University, Changchun 130021, P. R. China [3]School of Life Science, Jilin University, Changchun 130012, P R. China

出　　处：《Chemical Research in Chinese Universities》2015年第1期98-102,共5页高等学校化学研究（英文版）

基　　金：Supported by the National Natural Science Foundation of China（No.20772046）.

摘　　要：The aminopeptidase gene from thermophilic archaea Sulfolobustokodaii was cloned and expressed in Escherichia coli BL21 codon-plus（DE3）. To overexpress the aminopeptidase, the vector pET32a was constructed, in which the target gene was fused with the genes of histidine-tag and thioredoxin（Trx）. The expressed protein was purified using Ni^2＋-column affinity chromatography and ion exchange chromatography and cleft with enterokinase（EK） to obtain the purified aminopeptidase（ST1737）. The biochemical and enzymic properties of the expressed ST1737 were characterized. The results show that its optimal pH and temperature are 8 and 80 ℃, respectively. The half-life of ST1737（0.2 mg/mL） is about 85 h at 90 ℃, indicating that the enzyme exhibits an excellent thermostability. The activity of ST1737 could still maintain over 85% after its treatment at 25 ℃ in different buffers with a pH range of from 6.0 to 10.5 for 24 h, demonstrating that ST1737 is stable in neutral or slight alkali environment. The enzyme shows a high activity for the substrates such as unmodified peptide Asp-Ala, while the pNPC8 shows an optimal esterase substrate specificity. These results indicate that the enzyme is a bifunctional enzyme, and different from the aminopeptidase reported before.The aminopeptidase gene from thermophilic archaea Sulfolobustokodaii was cloned and expressed in Escherichia coli BL21 codon-plus（DE3）. To overexpress the aminopeptidase, the vector pET32a was constructed, in which the target gene was fused with the genes of histidine-tag and thioredoxin（Trx）. The expressed protein was purified using Ni^2＋-column affinity chromatography and ion exchange chromatography and cleft with enterokinase（EK） to obtain the purified aminopeptidase（ST1737）. The biochemical and enzymic properties of the expressed ST1737 were characterized. The results show that its optimal pH and temperature are 8 and 80 ℃, respectively. The half-life of ST1737（0.2 mg/mL） is about 85 h at 90 ℃, indicating that the enzyme exhibits an excellent thermostability. The activity of ST1737 could still maintain over 85% after its treatment at 25 ℃ in different buffers with a pH range of from 6.0 to 10.5 for 24 h, demonstrating that ST1737 is stable in neutral or slight alkali environment. The enzyme shows a high activity for the substrates such as unmodified peptide Asp-Ala, while the pNPC8 shows an optimal esterase substrate specificity. These results indicate that the enzyme is a bifunctional enzyme, and different from the aminopeptidase reported before.

关 键 词：AMINOPEPTIDASE Sulfolobustokodaii Thermostability 

分 类 号：Q814.1[生物学—生物工程] S858.285.3[农业科学—临床兽医学]