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| 创伤弧菌膦酸乙醛水解酶PhnX的重组表达、活性分析及晶体生长 |
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韩梦雪1,2,3, 刘长水1,2, 马庆军1,2,3
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1.中国科学院海洋研究所 实验海洋生物学重点实验室, 山东 青岛 266071;2.青岛海洋科技中心 海洋生物学与生物技术功能实验室, 山东 青岛 266237;3.中国科学院大学, 北京 100049
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| 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：10.11759/hykx20241210002 |
| 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);国家自然科学基金青年项目(41806203) |
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| Recombinant expression, enzyme activity, and crystallization of the Vibrio vulnificus phosphonoacetaldehyde hydrolase (PhnX) |
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HAN Mengxue1,2,3, LIU Changshui1,2, MA Qingjun1,2,3
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1.CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao 266237, China;3.University of Chinese Academy of Sciences, Beijing 100049, China
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| Abstract: |
| Phosphorus (P) is an essential nutrient for life. P limitation often occurs in the oceans; marine microorganisms can utilize inorganic phosphate (Pi) and organophosphorus compounds such as phosphonates as a P-acquisition strategy. 2-Aminoethylphosphonate is the most common phosphonate, and phosphonoacetaldehyde hydrolase (PhnX) is a key enzyme in converting phosphonate to Pi. However, the enzymatic properties of PhnX in marine bacteria have been rarely studied. In this effort, we successfully expressed and purified PhnX from Vibrio vulnificus (VvPhnX), which demonstrated significant enzymatic activity. Gel filtration experiments indicated VvPhnX to exist as a stable and soluble homomeric enzyme with a monomer molecular mass of ~29.5 kDa. Thermal shift experiments indicated the melting temperature to be ~37℃. After incubation at 20-70℃ for 1 h, the residual activity of VvPhnX declined with rising temperature. After incubation at 30℃ for 1 h, the enzyme activity declined by ~30%. At 40℃, the activity dropped by ~80% and was nearly lost after 1 h at ≥50℃; these results indicated the enzyme to have moderate thermal stability. The well-shaped crystals of the VvPhnX protein were obtained, and they were suitable for X-ray analysis. Overall, this research provides valuable insights into the biological function of PhnX and its role in the marine P cycle. |
| Key words: marine phosphorus cycle|organic phosphorus metabolism|2-Aminoethylphosphonate|phosphonoacetaldehyde hydrolase PhnX|Vibrio vulnificus |