| 摘要: |
| 磷是生命的必需元素。海洋中往往存在磷限制现象，海洋微生物采取了既能利用无机磷，又能利用膦酸盐等有机磷的磷获取策略。2-氨基乙基膦酸盐是最常见的膦酸盐，膦酸乙醛水解酶PhnX是将其转化为无机磷通路上的关键一环。然而，海洋细菌PhnX的酶学性质鲜有研究。本研究成功表达、纯化了具有酶活性的创伤弧菌PhnX，单体相对分子质量为29.5 kDa，该蛋白在溶液中以二聚体的形式存在。根据热稳定性实验，测定其Tm约为37 ℃，该酶在20-70℃下孵育1 h后，残余酶活随温度升高而下降，在30 ℃孵育1 h后酶活下降了约30%活性，40 ℃则下降了约80%的活性，在50 ℃及以上孵育1 h后酶活近乎消失，表明该酶具有中等热稳定性。本研究还获得了形状规则的蛋白晶体，为后续结构与工作机制研究奠定了基础。综上，本研究为理解海洋细菌PhnX生物学功能及其在海洋磷循环中的作用提供了有用信息。 |
| 关键词: 海洋磷循环，有机磷代谢，2-氨基乙基膦酸盐，膦酸乙醛水解酶PhnX，创伤弧菌 |
| DOI： |
| 分类号:Q93 |
| 基金项目:青岛市创新创业领军人才项目（No. 18-1-2-12-zhc） |
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| Recombinant expression, enzymatic activity and crystallization of phosphonoacetaldehyde hydrolase PhnX from Vibrio vulnificus |
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HanMengxue1,2,3, LiuChangshui1,2, MaQingjun1,2,3
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1.Institute of Oceanology, Chinese Academy of Sciences;2.Qingdao Marine Science and Technology Center;3.University of Chinese Academy of Sciences
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| Abstract: |
| Phosphorus is an essential nutrient for life. Phosphorus limitation often occurs in the ocean, and as a phosphorus acquisition strategy, marine microorganisms can utilize not only inorganic phosphate but also organophosphorus compounds such as phosphonates. 2-Aminoethylphosphonate is the most common phosphonate, and the phosphonoacetaldehyde hydrolase PhnX is a key enzyme involved in converting it to inorganic phosphate. However, the enzymatic properties and protein structure of PhnX in marine bacteria have been rarely studied. In this study, we successfully expressed and purified PhnX from Vibrio vulnificus (VvPhnX), which showed significant enzymatic activity. Based on gel filtration experiment, VvPhnX exists as a stable and soluble homodimeric enzyme with monomer molecular mass of approximately 29.5 kDa. Thermal shift experiment showed that the melting temperature of the enzyme was approximately 37 °C. After being incubated at 20–70 °C for 1 h, the residual activity of VvPhnX declined along with rising incubation temperature. After incubated at 30 °C for 1 hour, the enzyme activity was decreased by approximately 30%. At 40 °C, the activity was dropped by about 80%, and after 1 hour at equal to or more than 50 °C, the enzyme activity was nearly lost. This indicated that the enzyme has moderate thermal stability. The well-shaped protein crystals of VvPhnX were obtained, and those were suitable for further X-ray analysis. Overall, this research provides valuable insights into the biological function of phosphonoacetaldehyde hydrolase PhnX and its role in the marine phosphorus cycle. |
| Key words: Marine phosphorus cycle Organic phosphorus metabolism 2-Aminoethylphosphonate Phosphonoacetaldehyde hydrolase PhnX Vibrio vulnificus |