Wicka Monika, Wanarska Marta, Krajewska Ewelina, Pawlak-Szukalska Anna, Kur Józef, Cieśliński Hubert
Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland.
Acta Biochim Pol. 2016;63(1):117-125. doi: 10.18388/abp.2015_1074. Epub 2016 Jan 28.
An estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa. Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli strains for production of EstS9N (a two domain enzyme) and EstS9Δ (a one domain enzyme) proteins were constructed, respectively. Both recombinant proteins were successfully produced as inclusion bodies and then purified under denaturing conditions. However, because of the low enzymatic activity of the refolded EstS9Δ protein, only the EstS9N protein was further characterized. The purified and refolded EstS9N protein was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the butyrate (C4) ester. With p-nitrophenyl butyrate as the substrate, the enzyme displayed optimal activity at 35°C and pH 9.0. Additionally, the EstS9N esterase retained ~90% of its activity from 25-40°C and ~40% of its activity at 10°C. Moreover, analysis of its kinetic parameters (Km, kcat, kcat/Km) toward p-nitrophenyl butyrate determined at 15°C and 25°C confirmed that the EstS9 enzyme is cold-adapted. To the best of our knowledge, EstS9 is the third characterized cold-active GDSL-esterase and the first one confirmed to contain an autotransporter domain characteristic for enzymes secreted by the type V secretion system.
编码耐冷细菌假单胞菌属S9酯酶的estS9基因被克隆并测序。推导的序列显示该蛋白由636个氨基酸残基组成，分子量为69 kDa。进一步的氨基酸序列分析表明，EstS9酶含有以催化丝氨酸为中心的G-D-S-L基序、一个N端催化结构域和一个C端自转运结构域。分别构建了用于生产EstS9N（一种双结构域酶）和EstS9Δ（一种单结构域酶）蛋白的两种重组大肠杆菌菌株。两种重组蛋白均成功以包涵体形式产生，然后在变性条件下纯化。然而，由于复性后的EstS9Δ蛋白酶活性较低，仅对EstS9N蛋白进行了进一步表征。纯化并复性后的EstS9N蛋白对短链对硝基苯酯（C2-C8）具有活性，对丁酸（C4）酯具有最佳活性。以对硝基苯丁酸为底物时，该酶在35°C和pH 9.0下表现出最佳活性。此外，EstS9N酯酶在25-40°C下保留了约90%的活性，在10°C下保留了约40%的活性。此外，在15°C和25°C下测定其对硝基苯丁酸的动力学参数（Km、kcat、kcat/Km），证实EstS9酶是冷适应的。据我们所知，EstS9是第三个被表征的冷活性GDSL酯酶，也是第一个被证实含有V型分泌系统分泌酶特有的自转运结构域的酶。
