绿脓菌素促进铜绿假单胞菌NY3降解烃的作用机制
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摘要
绿脓菌素(Pyo)的分泌是铜绿假单胞菌NY3最为显著的特征之一,前期实验观察到该菌降解烃时, Pyo分泌量常与烃的降解效率成正相关性,但其在污染物降解中的作用尚未受到关注,且机制不清楚.以共存戊二酸为高分泌Pyo的体系和试剂级Pyo标准物为基础,研究了NY3菌降解烃时,Pyo对细胞内氧化还原酶及其比酶活力和胞外电子传递速率等因素的影响作用.结果表明,戊二酸能够明显提高Pyo的分泌量,相较于无戊二酸体系提高了86.6%,且随Pyo分泌量增加,NY3菌对十六烷去除率增加了16.29%;NY3菌分泌Pyo有利于提高其胞内烷烃氧化酶的活力,在菌体生长至72h、Pyo投加量分别为200,300μL时,相较于未投加Pyo体系,烷烃氧化酶比活力分别提高了121.8%和346.5%.同时Pyo存在下将胞外电子传递速率提高了近7倍,从而增加其对十六烷的降解速率.NY3菌分泌Pyo能通过提高胞内酶活性和胞外电子传递速率而促进NY3菌降解十六烷.
The secretion of pyocyanin(Pyo) is one of the most significant characteristics of Pseudomonas aeruginosa NY3. Previous experiments have observed that Pyo secretion is often positively correlated with hydrocarbon degradation efficiency, but its role in pollutant degradation has not been concerned, and the mechanism is unclear. Based on the coexistence of glutaric acid as a high-secreting Pyo system and reagent-grade Pyo standard, the effects of Pyo on intracellular redox enzyme activity, specific enzyme activity and extracellular electron transfer rate during hydrocarbon degradation by NY3 bacteria were studied. Glutaric acid could significantly increase the secretion of Pyo, which was 86.6% higher than that of non-glutaric acid system, and with the increase of Pyo secretion, the removal rate of hexadecane by NY3 bacteria increased by 16.29%. Pyo secretion by NY3 bacteria was beneficial to increase the activity of intracellular alkane oxidase. When the bacteria grew to 72 h and the dosage of Pyo was 200 L and 300 L, respectively, compared with that without Pyo system, alkane was removed by NY3 bacteria. The specific activity of hydrocarbon oxidase increased by 121.8% and 346.5% respectively. At the same time, in the presence of Pyo, the extracellular electron transfer rate was increased by nearly 7 times, which increased the degradation rate of hexadecane. Based on this, we propose that Pyo secretion by NY3 bacteria can promote the degradation of hexadecane by increasing the activity of intracellular enzymes and the rate of extracellular electron transfer.
The secretion of pyocyanin(Pyo) is one of the most significant characteristics of Pseudomonas aeruginosa NY3. Previous experiments have observed that Pyo secretion is often positively correlated with hydrocarbon degradation efficiency, but its role in pollutant degradation has not been concerned, and the mechanism is unclear. Based on the coexistence of glutaric acid as a high-secreting Pyo system and reagent-grade Pyo standard, the effects of Pyo on intracellular redox enzyme activity, specific enzyme activity and extracellular electron transfer rate during hydrocarbon degradation by NY3 bacteria were studied. Glutaric acid could significantly increase the secretion of Pyo, which was 86.6% higher than that of non-glutaric acid system, and with the increase of Pyo secretion, the removal rate of hexadecane by NY3 bacteria increased by 16.29%. Pyo secretion by NY3 bacteria was beneficial to increase the activity of intracellular alkane oxidase. When the bacteria grew to 72 h and the dosage of Pyo was 200 L and 300 L, respectively, compared with that without Pyo system, alkane was removed by NY3 bacteria. The specific activity of hydrocarbon oxidase increased by 121.8% and 346.5% respectively. At the same time, in the presence of Pyo, the extracellular electron transfer rate was increased by nearly 7 times, which increased the degradation rate of hexadecane. Based on this, we propose that Pyo secretion by NY3 bacteria can promote the degradation of hexadecane by increasing the activity of intracellular enzymes and the rate of extracellular electron transfer.
引文
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[17]樊晓宇,聂麦茜,徐鹏,等.左氧氟沙星对铜绿假单胞菌所产表面活性剂特性的影响作用[J].精细化工,2012,29(3):245-249.Fan X Y,Nie M Q,Xu P,et al.Effects of levofloxacin on the properties of surfactants produced by Pseudomonas aeruginosa[J].Fine Chemical Industry,2012,29(3):245-249.
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[19]陈帆,黄禹,叶慧.一种气相色谱定量方法的研究[J].温州师范学院学报(哲学社会科学版),2000,(6):38-39.Chen F,Huang Y,Ye H.Study on a Gas Chromatographic Quantitative Method[J].Journal of Wenzhou Normal University(Philosophy and Social Sciences Edition),2000,(6):38-39.
[20]Ewa Kaczorek,Karina Sa?ek,Urszula Guzik,et al.The impact of long-term contact of Achromobacter sp.4(2010)with diesel oil e Changes in biodegradation,surface properties and hexadecane monooxygenase activity[J].International Biodeterioration&Biodegradation,2013,78:7-16.
[21]Marion M.Bradford.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72:248-254.
[22]彭益强,李汉林.伴有辅酶原位再生的胞外酶耦合法制备(R)-苯基乙二醇[J].激光生物学报,2014,23(3):227-233.Peng Y Q,Li H L.Preparation of(R)-phenylethylene glycol by in situ regeneration of coenzymes[J].Journal of Laser Biology,2014,23(3):227-233.
[23]张小啸.土壤微生物降解石油组分的微观机理初探[D].北京:北京师范大学,2005.Zhang X X.A preliminary study on the micro-mechanism of soil microbial degradation of petroleum components[D].Beijing:Beijing Normal University,2005.
[24]白洁,崔爱玲,吕艳华.石油降解菌对石油烃的降解能力及影响因素研究[J].海洋湖沼通报,2007,(3):41-48.Bai J,Cui A L,Lu Y H.Study on the degradation ability of petroleum degrading bacteria to petroleum hydrocarbons and its influencing factors[J].Marine Lakes and Marshes Bulletin,2007,(3):41-48.
[25]张月梅.海洋低温石油降解菌的筛选及其降解机理[D].大连:大连工业大学,2010.Zhang Y M.Screening and Degradation Mechanism of Marine Low Temperature Petroleum Degrading Bacteria[D].Dalian:Dalian University of Technology,2010.
[26]Nie H Y.Evidences of extracellular abiotic degradation of hexadecane through free radical mechanism induced by the secreted phenazine compounds of P.aeruginosa NY3[J].Water Research,2018,(139):434-441.
[2]张博凡,熊鑫,韩卓,等.菌糠强化微生物降解石油污染土壤修复研究[J].中国环境科学,2019,39(3):1139-1146.Zhang B F,Xiong X,Han Z,et al.Study on microbial degradation of petroleum-contaminated soil remediation by fungus chaff[J].China Environmental Science,2019,39(3):1139-1146.
[3]潘声旺,雷志华,吴云霄,等.苏丹草根分泌物在有机氯农药降解过程中的作用[J].中国环境科学,2017,37(8):3072-3079.Pan S W,Lei Z H,Wu Y X et al.The role of Sudanese grass root exudates in the degradation of organochlorine pesticides[J].China Environmental Science,2017,37(8):3072-3079.
[4]赵鑫,韩妍,梁文,等.铜绿假单胞菌生物降解特性的研究进展[J].基因组学与应用生物学,2012,31(4):406-414.Zhao X,Han Y,Liang W,et al.Advances in biodegradability of Pseudomonas aeruginosa[J].Genomics and Applied Biology,2012,31(4):406-414.
[5]肖婷,聂麦茜,聂红云,等.草酸对铜绿假单胞菌NY3降解烃的作用研究[J].微生物学杂志,2018,38(2):70-76.Xiao T,Nie M Q,Nie H Y,et al.Study on the effect of oxalic acid on hydrocarbon degradation of Pseudomonas aeruginosa NY3[J].Journal of Microbiology,2018,38(2):70-76.
[6]陈延君,王红旗,王然,等.鼠李糖脂对微生物降解正十六烷以及细胞表面性质的影响[J].环境科学,2007,28(9):2117-2122.Chen Y J,Wang H Q,Wang R,et al.Effects of rhamnolipid on microbial degradation of n-hexadecane and cell surface properties[J].Environmental Science,2007,28(9):2117-2122.
[7]梁生康.鼠李糖脂生物表面活性剂对石油烃污染物生物降解影响的研究[D].青岛:中国海洋大学,2005.Liang S K.Study on the effect of rhamnolipid biosurfactant on the biodegradation of petroleum hydrocarbon pollutants[D].Qingdao:Dissertation of China Ocean University,2005.
[8]Krzysztof J,Reszka,Yunxia O’Malley,et al.Oxidation of pyocyanin,a cytotoxic product from pseudomonas aeruginosa,by microperoxidase11 and hydrogen peroxide[J].Free Radical Biology&Medicine,2004,36(11):1448-1459.
[9]O'Malley Y Q,Reszka K J,Spitz D R,et al.Pseudomonas aeruginosa pyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells[J].Am.J.Physiol.Lung Cell Mol.Physiol.,2004,87:94-103.
[10]Rada B,Lekstrom K,Damian S,et al.The Pseudomonas toxin pyocyanin inhibits the Dual oxidase-based antimicrobial system as it imposes oxidative stress on airway epithelial cells[J].Journal of Immunology,2008,181(7):4883-93.
[11]金宇,樊迪.自由基降解甲基橙溶液试验[J].江苏大学学报(自然科学版),2010,31(4):469-472.Jin Y,Fan D.Free radical degradation of methyl orange solution[J].Journal of Jiangsu University(Natural Science Edition),2010,31(4):469-472.
[12]张东峰.羟基自由基灭菌与降解有机环境污染物机理研究[D].秦皇岛:河北科技师范学院,2013.Zhang D F.Sterilization and degradation mechanism of organic pollutants by hydroxyl radicals[D].Qinhuangdao:Hebei Normal University of Science and Technology,2013.
[13]Price-Whelan A,Dietrich L E P,Newman D K.Pyocyanin alters redox homeostasis and carbon flux through central metabolic pathways in Pseudomonas aeruginosa PA14[J].Journal of Bacteriology,2007,189(17):6372-6381.
[14]聂红云.铜绿假单胞菌NY3胞外小分子活性物及其促进烃类降解的作用机制研究[D].西安:西安建筑科技大学,2017.Nie H Y.Pseudomonas aeruginosa NY3extracellular small molecule active substance and its mechanism of promoting hydrocarbon degradation[D].Xi'an:Xi'an University of Architecture and Technology,2017.
[15]Vandecasteele J P,Blanchet D,Tassin J P,et al.Enzymology of alkane degradation in pseudomonas aeruginosa[J].Acta Biotechnologica,2010,3(4):339-344.
[16]Nie M Q,Yin X H,Ren C Y,et al.Novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium Pseudomonas aeruginosa strain NY3[J].Biotechnology Advances,2010,28(5):635-643.
[17]樊晓宇,聂麦茜,徐鹏,等.左氧氟沙星对铜绿假单胞菌所产表面活性剂特性的影响作用[J].精细化工,2012,29(3):245-249.Fan X Y,Nie M Q,Xu P,et al.Effects of levofloxacin on the properties of surfactants produced by Pseudomonas aeruginosa[J].Fine Chemical Industry,2012,29(3):245-249.
[18]Grossowicz N,Peyuta hayat,Halpern Y S.Pyocyanine biosynthesis by Pseudomonas aeruginosa[J].J.gen.Microbial.,1957,16:576-583.
[19]陈帆,黄禹,叶慧.一种气相色谱定量方法的研究[J].温州师范学院学报(哲学社会科学版),2000,(6):38-39.Chen F,Huang Y,Ye H.Study on a Gas Chromatographic Quantitative Method[J].Journal of Wenzhou Normal University(Philosophy and Social Sciences Edition),2000,(6):38-39.
[20]Ewa Kaczorek,Karina Sa?ek,Urszula Guzik,et al.The impact of long-term contact of Achromobacter sp.4(2010)with diesel oil e Changes in biodegradation,surface properties and hexadecane monooxygenase activity[J].International Biodeterioration&Biodegradation,2013,78:7-16.
[21]Marion M.Bradford.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72:248-254.
[22]彭益强,李汉林.伴有辅酶原位再生的胞外酶耦合法制备(R)-苯基乙二醇[J].激光生物学报,2014,23(3):227-233.Peng Y Q,Li H L.Preparation of(R)-phenylethylene glycol by in situ regeneration of coenzymes[J].Journal of Laser Biology,2014,23(3):227-233.
[23]张小啸.土壤微生物降解石油组分的微观机理初探[D].北京:北京师范大学,2005.Zhang X X.A preliminary study on the micro-mechanism of soil microbial degradation of petroleum components[D].Beijing:Beijing Normal University,2005.
[24]白洁,崔爱玲,吕艳华.石油降解菌对石油烃的降解能力及影响因素研究[J].海洋湖沼通报,2007,(3):41-48.Bai J,Cui A L,Lu Y H.Study on the degradation ability of petroleum degrading bacteria to petroleum hydrocarbons and its influencing factors[J].Marine Lakes and Marshes Bulletin,2007,(3):41-48.
[25]张月梅.海洋低温石油降解菌的筛选及其降解机理[D].大连:大连工业大学,2010.Zhang Y M.Screening and Degradation Mechanism of Marine Low Temperature Petroleum Degrading Bacteria[D].Dalian:Dalian University of Technology,2010.
[26]Nie H Y.Evidences of extracellular abiotic degradation of hexadecane through free radical mechanism induced by the secreted phenazine compounds of P.aeruginosa NY3[J].Water Research,2018,(139):434-441.