嗜盐古菌Haloferax sp.D1227中超氧化物歧化酶功能鉴定及其增强细菌耐盐性的研究

Enhancement of bacterial salt tolerance by a newly identified superoxide dismutase from archaea Haloferax sp. D1227

【背景】细菌、酵母或植物来源的超氧化物歧化酶(Superoxide dismutase,SOD)编码基因在异源宿主中表达并提高宿主耐盐性的研究已有一些报道,其异源宿主也多为植物,而古菌来源的超氧化物歧化酶编码基因在细菌中成功表达并提高其耐盐性的研究尚无报道。【目的】寻找嗜盐古菌Haloferax sp.D1227中的超氧化物歧化酶编码基因并鉴定其功能,将其在4-硝基苯酚降解细菌Burkholderia sp.SJ98中表达,研究该古菌的超氧化物歧化酶对菌株SJ98耐盐性和降解4-硝基苯酚功能的影响。【方法】通过生物信息学方法寻找嗜盐古菌D1227中潜在的超氧化物歧化酶编码基因,利用表达载体p ET-28a和广泛宿主载体p BBR1MCS-2将其分别在E.coli BL21(DE3)和4-硝基苯酚的降解菌株SJ98中异源表达,检测细胞抽提液和纯化蛋白的超氧化物歧化酶比活力。分别以葡萄糖和4-硝基苯酚为碳源,在M9培养基和添加500 mmol/L Na Cl(Na Cl含量约3%)的M9培养基中分别培养细菌SJ98的重组菌株和空载体重组菌株,利用全自动生长曲线分析仪和高效液相色谱等方法检测重组菌株的生长能力和对4-硝基苯酚的降解能力。【结果】通过生物信息学分析,在嗜盐古菌D1227基因组中发现了潜在的超氧化物歧化酶编码基因sod A,其在E.coli BL21(DE3)和菌株SJ98中分别异源表达均具有超氧化物歧化酶活力[细胞抽提液的比活力分别为21.07±0.02 U/mg和84.56±0.16 U/mg,从BL21(DE3)菌株纯化的蛋白Sod AD1227比活力为179.46±3.43 U/mg]。在添加500 mmol/L Na Cl的M9培养基中培养时,以葡萄糖为碳源,重组菌株SJ98[p BBR-sod A]仍可正常生长,而空载体对照菌株SJ98[p BBR1MCS-2]几乎丧失了生长能力;以4-硝基苯酚为碳源,菌株SJ98[p BBR-sod A]保持了利用底物生长和降解底物的能力,而菌株SJ98[p BBR1MCS-2]的生长和降解能力几乎丧失。用软件Phyre2模拟分析Sod AD1227的单体结构,该蛋白拥有Fe/Mn-SOD家族的典型结构特征,推测其属于Fe/Mn-SOD家族。【结论】本研究为利用古菌SOD对细菌进行改造以适应高盐环境中降解有机污染物的应用提供了潜在的可行性。

[Background] Heterologous expression of many superoxide dismutases（SODs） from bacteria, yeast or plants have been reported to improve the salt tolerance of the host, but few from archaea. In particular, no SODs from archaea has been expressed in bacteria to improve their salt tolerance. [Objective] We are aiming to mine putative SODs encoding genes in archaea Haloferax sp. D1227, and to identify its biochemical function. It is also our intention to see possible enhancement of salt tolerance in para-nitrophenol utilizer Burkholderia sp. SJ98, by introducing the newly identified SOD. [Methods] The putative SOD encoding gene from strain D1227 was mined by bioinformatics analysis before its heterologous expression in E. coli BL21（DE3） and strain SJ98, which was further purified through AKTA purifier system. The specific activity of cell extracts and purified enzyme were measured by a spectrophotometry. Cells of strain SJ98 with the SOD gene and with vector only were cultured in M9 medium（with 0 and 500 mmol/L Na Cl） with glucose or PNP as carbon source, respectively. Subsequently, the growth and degradation of these strains were detected by automatic growth curve analyzer and high-performance liquid chromatography. [Results] A putative SOD encoding gene from Haloferax sp. D1227 was found and designated sod A. Sod AD1227 heterologous expressed in both E. coli BL21（DE3） and strain SJ98 exhibited SOD activity（with specific activities of 21.07±0.02 U/mg and 84.56±0.16 U/mg respectively in cell extracts）. The specific activity of purified protein from E. coli was 179.46±3.43 U/mg. Strain SJ98[p BBR-sod A] grew well in M9 containing 500 mmol/L Na Cl with glucose as carbon source, while strain SJ98[p BBR1 MCS-2] almost lost its growth ability. When para-nitrophenot（PNP） was used as carbon source, strain SJ98[p BBR-sod A] still had a normal growth with a proper PNP degradation ability, while the growth and degradation ability of strain SJ98[p BBR1 MCS-2] was almost lost. The structural analysis of Sod AD1227 simulated by Phyre2 showed that Sod AD1227 has the typical structural characteristics of the Fe/Mn-SOD family. [Conclusion] This study provides a potential feasibility for the use of archaeal SODs in transforming bacteria to adapt to the degradation of organic pollutants in high salinity environment.