張 波,聶麥茜,2*,聶紅云,2,田曉婷,喬 琦

綠膿菌素促進(jìn)銅綠假單胞菌NY3降解烴的作用機(jī)制

張 波1,聶麥茜1,2*,聶紅云1,2,田曉婷1,喬 琦1

(1.西安建筑科技大學(xué)環(huán)境與市政工程學(xué)院,陜西 西安 710055；2.西安建筑科技大學(xué),陜西省膜分離重點(diǎn)實(shí)驗(yàn)室,陜西 西安 710055)

綠膿菌素(Pyo)的分泌是銅綠假單胞菌NY3最為顯著的特征之一,前期實(shí)驗(yàn)觀察到該菌降解烴時(shí), Pyo分泌量常與烴的降解效率成正相關(guān)性,但其在污染物降解中的作用尚未受到關(guān)注,且機(jī)制不清楚.以共存戊二酸為高分泌Pyo的體系和試劑級(jí)Pyo標(biāo)準(zhǔn)物為基礎(chǔ),研究了NY3菌降解烴時(shí),Pyo對(duì)細(xì)胞內(nèi)氧化還原酶及其比酶活力和胞外電子傳遞速率等因素的影響作用.結(jié)果表明,戊二酸能夠明顯提高Pyo的分泌量,相較于無(wú)戊二酸體系提高了86.6%,且隨Pyo分泌量增加,NY3菌對(duì)十六烷去除率增加了16.29%;NY3菌分泌Pyo有利于提高其胞內(nèi)烷烴氧化酶的活力,在菌體生長(zhǎng)至72h、Pyo投加量分別為200,300μL時(shí),相較于未投加Pyo體系,烷烴氧化酶比活力分別提高了121.8%和346.5%.同時(shí)Pyo存在下將胞外電子傳遞速率提高了近7倍,從而增加其對(duì)十六烷的降解速率.NY3菌分泌Pyo能通過(guò)提高胞內(nèi)酶活性和胞外電子傳遞速率而促進(jìn)NY3菌降解十六烷.

銅綠假單胞菌NY3；綠膿菌素(pyo)；烴類；烷烴氧化酶；戊二酸

烷烴是原油的主要成分,烷烴的降解是自然界普遍存在的現(xiàn)象.許多微生物,包括原核生物和真核生物,都能利用烷烴作為碳源和能源[1].目前已有研究利用菌糠強(qiáng)化微生物對(duì)石油烴污染進(jìn)行修復(fù)[2],也有報(bào)道指出,有生物胞外分泌物可促進(jìn)有機(jī)物的生物降解過(guò)程[3].銅綠假單胞菌NY3(NY3)不僅能高效降解烴類,且可代謝的烴種類多、碳鏈長(zhǎng)度范圍廣[2].該菌突出的烴降解能力與其胞外分泌的多種活性小分子化合物密切相關(guān)[3].如胞外分泌的鼠李糖脂,能明顯提其對(duì)烴類的降解效率,且其促進(jìn)機(jī)理也被廣泛論證[4-5].而作為NY3的另一特征分泌物-綠膿菌素(Pyo),其對(duì)烴的降解作用研究較少.目前關(guān)于Pyo的研究主要集中在醫(yī)學(xué)領(lǐng)域,研究表明,Pyo能自由穿透生物膜,參與氧化還原反應(yīng),產(chǎn)生活性氧[6];且可與細(xì)胞外的還原性輔酶Ⅰ(NADH)、還原型谷胱甘肽(GSH),同時(shí)產(chǎn)生氧自由基[[7-8],自由基對(duì)于有機(jī)物的降解有著非常重要的作用[9-10].也有人報(bào)道,在銅綠假單胞菌代謝葡萄糖的過(guò)程中,Pyo能夠改變自身細(xì)胞內(nèi)的氧化還原狀態(tài),加速其對(duì)碳源的代謝過(guò)程[11].烴屬于有機(jī)物,亦屬于碳源,在銅綠假單胞菌對(duì)其降解過(guò)程中,會(huì)分同步泌Pyo,其分泌量與代謝條件、以及烴的降解速率密切相關(guān).實(shí)驗(yàn)室前期研究NY3菌代謝烴時(shí)發(fā)現(xiàn),NY3在快速代謝烴類時(shí),會(huì)分泌大量的Pyo等吩嗪類物質(zhì);胞外液中的吩嗪類物質(zhì)(包括Pyo)可與胞外液中的NADH、GSH等反應(yīng),加速烴類的胞外降解速率.共存碳源戊二酸可加速NY3菌降解十六烷的過(guò)程,且可使胞外液中Pyo的分泌量增加[12].然而,尚不清楚胞外液中的Pyo在促進(jìn)烴類在NY3菌的胞外降解過(guò)程中,對(duì)胞內(nèi)降解有何作用,其作用機(jī)理也有待揭示.目前僅有報(bào)道中提及以烷烴為碳源時(shí)烷烴降解酶之一的醛去氫酶相較于葡萄糖、琥珀酸或丙二酸為碳源時(shí)更加有活性[13].本文基于前期的研究結(jié)果,選擇戊二酸為NY3菌高分泌Pyo的共代謝碳源,同時(shí)借助純品Pyo,研究其對(duì)烴類降解關(guān)鍵酶與降解體系電子傳遞速率的影響,以期揭示Pyo在NY3菌代謝烴過(guò)程中作用機(jī)制.

1 材料與方法

1.1 實(shí)驗(yàn)材料

1.1.1 菌種來(lái)源 銅綠假單胞菌NY3,經(jīng)實(shí)驗(yàn)室分離并鑒定[14].

1.1.2 試劑與培養(yǎng)基 無(wú)機(jī)鹽培養(yǎng)基的制備方法參照前期文獻(xiàn)[15],其組成主要包括(每升含量): 1.0gNH4NO3,25mL 磷酸鹽緩沖液,1mL微量元素,0.5mL 1mol/L MgSO4?7H2O,0.1mL 1mol/L CaCl2?2H2O.將無(wú)機(jī)鹽pH值調(diào)至7.5,并于121℃滅菌30min后,備用.

種子液培養(yǎng)基制備:牛肉膏3.0g,蛋白胨10.0g, NaCl 5.0g,調(diào)pH=7.5,溶解于1000mL蒸餾水中, 121℃,滅菌30min后,備用.

1.2 實(shí)驗(yàn)方法

1.2.1 種子液制備 無(wú)菌操作條件下,NY3接種于100mL種子液培養(yǎng)基,在恒溫振蕩器中150r/min, 30℃培養(yǎng)24h,OD600nm調(diào)至1.6±0.05,備用.

1.2.2 戊二酸對(duì)NY3分泌Pyo的影響 從文獻(xiàn)[16]結(jié)果看,戊二酸可促進(jìn)銅綠假單胞菌分泌Pyo.取18mL無(wú)機(jī)鹽培養(yǎng)基于50mL錐形瓶中,外加30μL十六烷,接種10%(/),加戊二酸,濃度達(dá)到0.1mol/L,以十六烷為唯一碳源的培養(yǎng)體系為對(duì)照.150r/min, 30℃恒溫培養(yǎng)16h,測(cè)培養(yǎng)液pH值、OD600nm,并用正己烷萃取其中剩余的十六烷.以正十二烷為內(nèi)標(biāo)[17],用氣相色譜測(cè)定十六烷量,并計(jì)算降解率.同時(shí)各用10mL氯仿萃取5mL降解液,收集氯仿相,用5mL 0.2mol/L HCl反萃取氯仿相,測(cè)HCl相OD378nm值,按照純品Pyo的標(biāo)準(zhǔn)曲線計(jì)算培養(yǎng)液中Pyo的分泌量(mg/L).

1.2.3 NY3生長(zhǎng)產(chǎn)Pyo及其提取 加戊二酸于90mL無(wú)機(jī)鹽培養(yǎng)基中,濃度達(dá)0.1mol/L,NY3菌接種10%(/).150r/min,30℃恒溫培養(yǎng)24h.按照1.2.2中方法重復(fù)操作2次萃取Pyo.將Pyo收集到氯仿相,揮發(fā)溶劑.用蒸餾水溶解,并調(diào)OD378nm至1.032 (22.51mg/L),儲(chǔ)于4℃冰箱,備用.

1.2.4 高分泌Pyo對(duì)NY3菌氧化還原酶比活力的影響 按照1.2.2中方法投加十六烷和NY3種子液,戊二酸投加至濃度為0.1mol/L,以未加戊二酸為對(duì)照.150r/min,30℃恒溫培養(yǎng),分別于10,14,18,24,48, 72h采樣,測(cè)定OD600nm的吸光度,用以表示生長(zhǎng)體系的菌量.用正己烷萃取剩余十六烷,十二烷為內(nèi)標(biāo),氣相色譜測(cè)定十六烷含量,并計(jì)算降解率.同時(shí)10000r/ min離心收獲10,14,18,24,48,72h等時(shí)刻100mL培養(yǎng)液中的菌體,用pH=7.2磷酸鹽緩沖液洗滌菌體3次,并制備成菌懸液.置于0℃冰水浴中,用超聲波細(xì)胞破碎儀間歇處理20次,每次1min,功率為450W.破碎后,4℃,10000r/min離心10min,上清液為粗酶液.粗酶液的氧化還原反應(yīng)酶活性以NADH的反應(yīng)速率表示[18].取3mL pH=7.2的磷酸鹽緩沖液于10mL離心管中,加2mL粗酶液,30μL 26.2mmol/L NADH水溶液,加入200μL(22.51mg/L)的Pyo,以未加Pyo為對(duì)照,測(cè)定1min內(nèi)OD340nm處吸光度變化量,每隔10s記錄一次.每個(gè)樣品平行3個(gè)實(shí)驗(yàn).并測(cè)定粗酶液中蛋白濃度[19].按下式計(jì)算粗酶液烷烴氧化酶比活力[20].

式中:為烷烴氧化酶比活力,U/mg;t為反應(yīng)液總體積,mL;s為粗酶液體積,mL;為NADH摩爾消光系數(shù),6.22L/(mmol×cm);為比色皿厚度,cm;D/D為每min吸光度變化值,min-1;pr為蛋白濃度,mg/L.

1.2.5 Pyo對(duì)NY3菌粗酶液催化氧化十六烷效率的影響 取90mL無(wú)機(jī)鹽于250mL錐形瓶中,加入150μL十二烷,NY3菌接種量10%(/),150r/min, 30℃恒溫培養(yǎng)24,48,72h離心收集菌體,并按上述方法洗滌和制備粗酶液.取3mL pH=7.2的磷酸鹽緩沖液于10mL反應(yīng)瓶中,加2mL粗酶液,30μL 26.2mmol/L NADH溶液,5.0μL十六烷,分別加200,300μL上述Pyo(22.51mg/L)溶液,以未加Pyo為對(duì)照.按照1.2.4測(cè)定并計(jì)算菌體氧化還原酶比活力.反應(yīng)15min后,用上述方法萃取并測(cè)定剩余的十六烷含量,并計(jì)算降解率.

1.2.6 高分泌Pyo對(duì)NY3菌降解十六烷體系電子傳遞速率的影響 采用CHI660A電化學(xué)系統(tǒng)(上海CHI公司)在-0.8~+0.8V電位范圍間記錄其循環(huán)伏安(CV)曲線.其中,降解體系樣品制備方法為為直接吸取10mL降解體系;上清液樣品制備方法為取10mL降解體系于12000,4℃離心20min,將所得上清液過(guò)0.22mm濾膜,除去未離心掉的菌體,所得液體即為上清樣品;菌體制樣方法為將上清制樣過(guò)程中離心所得菌體以無(wú)機(jī)鹽洗滌3次,最后重懸于10mL無(wú)機(jī)鹽中,即為菌體的電化學(xué)檢測(cè)樣品.電化學(xué)檢測(cè)采用三電極體系,其中SCE為參比電極,鉑絲為對(duì)電極,GCE或Ft-SWNTs-CTAB/GCE 為工作電極.

2 結(jié)果與討論

2.1 戊二酸作為共存碳源對(duì)NY3菌分泌Pyo的影響

NY3以十六烷(C16)為碳源生長(zhǎng)的發(fā)酵液顏色呈淺綠色,C16+戊二酸為碳源的發(fā)酵液為的深綠色,該顏色屬于典型的Pyo顏色.因此,基于胞外液的顏色,可初步判斷外加戊二酸能使NY3菌產(chǎn)較多的Pyo.為了進(jìn)一步驗(yàn)證戊二酸的影響作用,分別測(cè)定NY3菌生長(zhǎng)體系中OD600nm、pH值、Pyo濃度及生長(zhǎng)單位菌體(以O(shè)D600nm表示)對(duì)十六烷去除率等指標(biāo),結(jié)果如表1所示.由表1可看出,在以十六烷為碳源生長(zhǎng)的NY3菌培養(yǎng)液中,投加戊二酸可促進(jìn)菌體的生長(zhǎng),同時(shí)提高Pyo的分泌量.一般來(lái)說(shuō),菌體生長(zhǎng)量越高,必然導(dǎo)致碳源(十六烷)利用率提高.為了對(duì)比不同生長(zhǎng)體系菌體對(duì)十六烷的代謝活性,以十六烷去除率除以培養(yǎng)液的OD600nm,表示單位菌體對(duì)十六烷去除率.結(jié)果表明,外加戊二酸對(duì)NY3菌體生長(zhǎng)、Pyo分泌量、十六烷去除率以及單位菌體去除的十六烷的效率均有明顯提高.因此,戊二酸代謝促進(jìn)了NY3菌細(xì)胞的生長(zhǎng)以及胞外Pyo的分泌,但其促進(jìn)機(jī)制還有待進(jìn)一步研究.

表1 共存碳源存在下NY3菌生長(zhǎng)16h培養(yǎng)液中相關(guān)指標(biāo)的測(cè)定結(jié)果

注:碳源十六烷投加量30μL;戊二酸投加量:濃度達(dá)到0.1mol/L.

2.2 高分泌Pyo對(duì)NY3菌細(xì)胞中烴氧化酶活力的影響

將不同生長(zhǎng)階段的NY3菌細(xì)胞破碎后,測(cè)定所獲粗酶液中具有十六烷氧化能力的烴氧化酶活力及不同時(shí)間點(diǎn)的Pyo產(chǎn)量,結(jié)果如圖1所示.戊二酸存在的體系中菌體的烴氧化酶活力力均高于對(duì)照體系,投加戊二酸的體系中Pyo的產(chǎn)量均高于無(wú)戊二酸體系,尤其是在14h后兩者差距更大.而酶活測(cè)定時(shí)菌體經(jīng)過(guò)了離心、重懸、破碎處理.粗酶液中并無(wú)Pyo的存在,因此酶活性的高低取決于酶蛋白的含量.綜上可知,戊二酸在促進(jìn)菌體分泌Pyo的同時(shí)還使得烴氧化酶的合成增加.即戊二酸的存在能夠使Pyo處于高分泌狀態(tài),Pyo可使NY3菌的氧化還原酶活性活力提高,說(shuō)明戊二酸可通過(guò)直接影響Pyo的分泌從而間接影響NY3菌對(duì)十六烷的降解率.研究表明,微生物是先將石油烴組分運(yùn)移至細(xì)胞內(nèi)部,再進(jìn)行細(xì)胞內(nèi)生化降解,則胞內(nèi)烷烴氧化酶的數(shù)量則直接會(huì)影響烷烴的降解[21].

2.3 Pyo對(duì)NY3菌細(xì)胞粗酶液中烴氧化酶比活力的影響

表2 Pyo對(duì)NY3菌粗酶液催化氧化十六烷效率的影響作用

注:所有所投加的Pyo濃度均為22.51mg/L.

測(cè)定不同濃度的純品Pyo對(duì)NY3菌粗酶液中烴氧化酶比活力及其對(duì)十六烷降解率,結(jié)果如表2所示.對(duì)于24h收獲的菌體,加入200,300μL Pyo (22.51mg/L),與未加Pyo相比,氧化還原酶比活力分別提高了0.14,0.25U/mg,15min內(nèi)粗酶液對(duì)十六烷去除率增加了0.6%、5%.對(duì)于48h和72h所收獲菌體的粗酶液,加Pyo對(duì)于氧化還原酶比活力和十六烷的去除率均有提高,提高幅度較24h菌體粗酶液小.因此,Pyo不僅可提高NY3菌粗酶的液氧化還原酶比活力,也能提高粗酶液對(duì)烷烴的去除率,說(shuō)明Pyo在NY3菌體降解烴類物質(zhì)時(shí),參與了氧化還原酶的電子傳遞系統(tǒng),對(duì)提高酶的活性是有利的.相較于其他研究[22-23]中通過(guò)調(diào)節(jié)微生物生長(zhǎng)過(guò)程中的其他物理因素影響菌體的生長(zhǎng)從而影響降解效率,Pyo是直接作用于參與氧化烷烴的酶,使其比活力提高,進(jìn)而加快烷烴降解.

2.4 Pyo對(duì)NY3菌降解烴體系中電子傳遞速率的影響

由圖2(a)可知,當(dāng)以十六烷為唯一碳源時(shí)(對(duì)照組),NY3菌降解十六烷反應(yīng)液的C-V曲線中存在一個(gè)氧化還原電對(duì)和一個(gè)氧化電位.當(dāng)戊二酸共存時(shí),NY3菌降解十六烷反應(yīng)液的C-V曲線較十六烷為唯一碳源時(shí)多出一個(gè)氧化還原電對(duì),且相同電壓下體系的電流加大,電子傳遞速度加快.由此可知,戊二酸共存可使NY3菌降解十六烷體系分泌一種新的氧化還原物質(zhì),且可加速體系的電子傳遞速度,從而促進(jìn)了十六烷的降解.由圖2(b)可知,外加純品Pyo時(shí),在相同的電壓條件下的響應(yīng)電流要明顯大于未加Pyo的體系,即Pyo可使體系的電子傳遞速率增大.上述結(jié)果表明,戊二酸的存在可促進(jìn)Pyo的分泌從而導(dǎo)致降解體系內(nèi)的電子傳遞速率,進(jìn)而提高了對(duì)十六烷的降解效率

2.5 Pyo對(duì)NY3菌降解十六烷影響機(jī)制的推測(cè)

基于本研究的上述結(jié)果以及前期工作[24-26],本文推測(cè)Pyo參與NY3菌降解十六烷的作用機(jī)制如圖3所示.

圖3 Pyo影響NY3菌降解十六烷的過(guò)程模型

據(jù)圖3可知,NY3菌降解烷烴的過(guò)程中同步分泌Pyo,對(duì)于菌體細(xì)胞內(nèi)代謝過(guò)程來(lái)說(shuō),Pyo可使烴氧化酶處于高活性狀態(tài),提高烴氧化酶活力及氧化酶比活力,從而加速烴類物質(zhì)的酶促降解過(guò)程.對(duì)于分泌到胞外Pyo來(lái)說(shuō),其可作為電子穿梭體,加速電子在有機(jī)物于氧氣之間的傳遞,從而增加降解體系的電子傳遞.通過(guò)這兩方的總和作用,Pyo加速了NY3菌對(duì)烴類的降解過(guò)程.

3 結(jié)論

3.1 投加戊二酸能提高NY3菌對(duì)Pyo的分泌,相較于無(wú)戊二酸體系提高了86.6%,同時(shí)可提高NY3菌的細(xì)胞生長(zhǎng)量和單位菌體對(duì)十六烷的降解活性,16h時(shí)十六烷去除率增加了16.29%.

3.2 Pyo能提高NY3菌胞內(nèi)烴氧化酶的合成及其比活力,增加其對(duì)十六烷的降解速率,在菌體生長(zhǎng)至72h、Pyo投加量分別為200,300μL時(shí),相較于未投加Pyo體系,烷烴氧化酶比活力分別提高了121.8%和346.5%;Pyo亦可通提高降解體系電子傳遞速率,提高幅度達(dá)7倍之多.

3.3 Pyo促進(jìn)NY3菌降解十六烷的機(jī)制是通過(guò)對(duì)胞內(nèi)關(guān)鍵酶活性的促進(jìn)與對(duì)胞外電子傳遞速率的同時(shí)進(jìn)行.

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The effects of pyocyanin on alkanes degradation byNY3.

ZHANG Bo1, NIE Mai-qian1,2*, NIE Hong-yun1,2, TIAN Xiao-ting1, QIAO Qi1

(1.School of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China；2.Shaanxi Key Laboratory of Membrane Separation, Xi’an University of Architecture and Technology, Xi’an 710055, China)., 2019,39(7)：3088~3093

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 72h and the dosage of Pyo was 200L and 300L, 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.

NY3；pyocyanine pyo；hydrocarbons；alkane oxidase；glutaric acid

X172

A

1000-6923(2019)07-3088-06

張 波(1993-),男,陜西延安人,西安建筑科技大學(xué)碩士研究生,從事持久性有機(jī)污染物的生物降解技術(shù)研究.

2018-12-05

陜西省科技廳重點(diǎn)產(chǎn)業(yè)創(chuàng)新鏈項(xiàng)目(2019ZDLSF05-04);中國(guó)博士后科學(xué)基金(2018M633479);陜西省教育廳科研計(jì)劃項(xiàng)目(18JK0449)

*責(zé)任作者, 教授, niemaiqian@xauat.edu.cn