第34卷第9期西北農(nóng)林科技大學(xué)學(xué)報(bào)(自然科學(xué)版)2006年9月 Vol. 34 No. 9 Jour. of Northwest Sci-Tech Univ. of Agri. and For. (Nat. Sci. Ed.)Sep.2006乙醇對(duì)土壤轉(zhuǎn)化酶活性的影響王聰穎,和文祥,何敏超(西北農(nóng)林科技大學(xué)資源環(huán)境學(xué)院,陜西楊凌712100[摘要]研究了不同濃度乙醇對(duì)土壤轉(zhuǎn)化酶活性的影響以及不同底物(蔗糖)濃度對(duì)乙醇與轉(zhuǎn)化酶活性關(guān)系的影響。結(jié)果表明,乙醇可明顯抑制土壤轉(zhuǎn)化酶活性,其濃度與土壤轉(zhuǎn)化酶活性呈顯著或極顯著相關(guān),其作用方式為直接抑制乙醇輕度污染時(shí)的生態(tài)劑量ED1值為29.47%~35.27%,表明乙醇是一種毒性不強(qiáng)的有機(jī)污染物;底物濃度可放大乙醇與土壤轉(zhuǎn)化酶活性之間的關(guān)系;乙醇加導(dǎo)致土壤轉(zhuǎn)化酶的Km和vm值小,揭示其作用機(jī)理為完全抑制作用,包括競(jìng)爭(zhēng)性和非競(jìng)爭(zhēng)性抑制作用。[關(guān)鍵詞]乙醇;轉(zhuǎn)化酶活性;動(dòng)力學(xué);作用機(jī)理[中圖分類號(hào)]S154.2[文獻(xiàn)標(biāo)識(shí)碼]A[文章編號(hào)]1671-93872006)09-0125-05乙醇作為一種有機(jī)污染物,其來源非常廣泛,可染的程度;甲苯進(jìn)入土壤后會(huì)顯著提高土壤脲酶的大量存在于一些生活和工業(yè)廢水中在土壤有機(jī)物活性,甲醇可增加大多數(shù)土壤脲酶的活性等但至今質(zhì)(秸稈、有機(jī)肥料等)的轉(zhuǎn)化過程中也會(huì)形成乙醇。有關(guān)乙醇對(duì)土壤酶活性的影響鮮見報(bào)道為此本試此外,某些農(nóng)藥原藥在使用時(shí)也需要加人乙醇。這些驗(yàn)研究了乙醇對(duì)土壤轉(zhuǎn)化酶活性的影響,以期為環(huán)乙醇最終都會(huì)進(jìn)入土壤生態(tài)系統(tǒng),對(duì)土壤性質(zhì)包括境保護(hù)提供重要依據(jù)。其中生長(zhǎng)的微生物等產(chǎn)生影響因此,乙醇生態(tài)毒理效應(yīng)的研究在理論和實(shí)踐上具有十分重要的意義。1材料與方法土壤酶是土壤的重要組成部分,在物質(zhì)轉(zhuǎn)1.1供試土樣化能量代謝和污染物降解等方面發(fā)揮著重要作用。土樣于2002-03采自西北農(nóng)林科技大學(xué)從近年來,國(guó)內(nèi)外許多學(xué)者3研究了外源污染物(主1979年開始的土壤長(zhǎng)期肥料定位試驗(yàn)地(培肥方案要包括重金屬、農(nóng)藥及其他有機(jī)物質(zhì))對(duì)土壤酶活性見表1)。采樣時(shí)先去除0~5cm的表土,再取5~20的影響,并且取得了較大的進(jìn)展,如發(fā)現(xiàn)采用多個(gè)土cm土層的土樣,混勻風(fēng)干后,過1mm篩備用。壤酶構(gòu)建的酶系統(tǒng)可較為敏感地表征土壤重金屬污表1供試土壤的培肥方案 Table, 1 Design of long-term fertilizer experiment kg/hm2'土樣編號(hào) Soil samples處理尿素過磷酸鈣秸稈 Treatment Urea Ca(H2PO)2肥 Straw Manure無肥o- o-fertilizer0000化肥 Chemical fertilizer52500高 High straw4505375000450525375001.2乙醇對(duì)土壤轉(zhuǎn)化酶活性的影響基水楊酸比色法0測(cè)定土壤轉(zhuǎn)化酶活性。土壤轉(zhuǎn)化取5.00g土樣,向其中加入5滴甲苯后,再加1酶活性單位定義為每小時(shí)在1g土樣中由蔗糖轉(zhuǎn)mL不同體積分?jǐn)?shù)(0%,5%,10%,25%,50%,化成葡萄糖的毫克數(shù)。每處理重復(fù)3次,并設(shè)無底物100%)的乙醇溶液混勻后靜置30min,最后加15和無土壤處理為對(duì)照。mL濃度為80g/L的蔗糖溶液和5mL磷酸緩沖液1.3底物濃度對(duì)乙醇與土壤轉(zhuǎn)化酶活性關(guān)系的影響(pH5.5),搖勻,37C培養(yǎng)5h后取樣。用3,5-二硝以1號(hào)土樣為例,實(shí)驗(yàn)方法同1.2.1,底物蔗糖[收稿日期]2005-10-08[基金項(xiàng)目國(guó)家自然科學(xué)基金項(xiàng)目(4030102)國(guó)家973重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃子項(xiàng)目(2004CB418506)[作者簡(jiǎn)介]王聰穎(1981),女[通訊】王聰(1=:,西長(zhǎng)人,在讀硬士,主要從事土壤生物化學(xué)和生態(tài)毒理研究e-malwxh968163.co要從事土壤生態(tài)毒理研究者]和文(19男陜西黃龍126西北農(nóng)林科技大學(xué)學(xué)報(bào)(自然科學(xué)版)第34卷的濃度分別為:0.010,0.025,0.050和0.100應(yīng)初始和t時(shí)間時(shí)的底物濃度。mol/L,取樣時(shí)間為3hS1.4乙醇作用時(shí)間對(duì)土壤轉(zhuǎn)化酶活性的影響將不同時(shí)間的n和(S-[S])值進(jìn)實(shí)驗(yàn)方法同1.2.1,乙醇濃度為體積分?jǐn)?shù)50%,行線性擬合,即可求得Km和Vm值靜置時(shí)間分別為:0,15,30,60,120和240min。1.5.2對(duì)土壤轉(zhuǎn)化酶促反應(yīng)速度常數(shù)k的影響1.5乙醇對(duì)土壤轉(zhuǎn)化酶動(dòng)力學(xué)特征的影響以1號(hào)土樣為例來研究乙醇對(duì)土壤轉(zhuǎn)化酶動(dòng)力k=nvv,式中,V和V分別為酶促反應(yīng)產(chǎn)學(xué)特征的影響。生的最大葡萄糖量和t時(shí)間產(chǎn)生的葡萄糖量。1.5.1對(duì)土壤轉(zhuǎn)酶動(dòng)力學(xué)參數(shù)Km和的影2結(jié)果與分析響經(jīng)典米氏方程: V.= X [S]2.1乙醇對(duì)土壤轉(zhuǎn)化酶活性的影響 Km+[]結(jié)果顯示,(1)土壤轉(zhuǎn)化酶活性大小次序?yàn)?1經(jīng)數(shù)學(xué)變換為:號(hào)土樣<2號(hào)土樣<3號(hào)土樣<4號(hào)土樣,其中1和 In S-Kx ([S.]-[S])+4號(hào)土樣的土壤轉(zhuǎn)化酶活性分別為0.77和1.08mg/(g·h),二者相差40.26%(表2)。這表明土壤式中,t為酶促反應(yīng)時(shí)間;[S]和[S]分別為酶促反轉(zhuǎn)化酶活性與土壤肥力水平密切相關(guān)。表2不同濃度乙醇對(duì)土壤轉(zhuǎn)化酶活性的影響 Table 2 Invertase activity in soils tested under different concentrations of ethanol mg/)土樣編號(hào)乙醇體積分?jǐn)?shù)/% Concentration of ethanol Soil sample051025501000.770.760.70.670.610.5720.920.810.790.800.740.6631.071.130.950.850.740.821.081.110.980.9440.880.83注:100%的乙醇指的是分析純級(jí)乙醇 Note: 100% means that ethanol is analytical grade.(2)與對(duì)照相比,除3號(hào)和4號(hào)土樣中加入5%結(jié)果類似。的乙醇使土壤轉(zhuǎn)化酶活性升高外,其余濃度乙醇的(3)當(dāng)乙醇濃度為5%~100%時(shí),隨乙醇濃度加入均導(dǎo)致土壤轉(zhuǎn)化酶活性降低,當(dāng)乙醇濃度達(dá)到增加,土壤轉(zhuǎn)化酶活性總體上持續(xù)減小(表2)為此100%時(shí),1,2,3,4號(hào)土樣的土壤酶活性與各自的乙采用 Speir等[12提出的U=A/(1+B×C)模型,對(duì)醇體積分?jǐn)?shù)為0時(shí)的酶活性相比,降幅分別為本研究中的乙醇濃度(C)與轉(zhuǎn)化酶活性(U)的關(guān)系25.97%,28.26%,23.36%和23.15%(表2)這與進(jìn)行擬合,結(jié)果見表3。 Sannino等研究發(fā)現(xiàn)的甲醇對(duì)土壤轉(zhuǎn)化酶的影響表3土壤轉(zhuǎn)化酶活性與乙醇濃度關(guān)系的擬合結(jié)果 Table Regression equation between soil invertase activity and ethanol concentration土樣編號(hào)參數(shù) Parameters Soil sampleAB ED10/%0.7520.3770.95629.470.8570.3150.90235.271.04510.4710.7781.0630.3400.90936.28注:自由度n-2=4.0=0.81,ra.01=0.917;表示顯著相關(guān);表示極顯著相關(guān)表5同 Note: Freedom n-2= 4,. =0. 811,. 0. 917. remarkable correlation, :significant remarkable correlation. Table 5 is the由表3可知,除3號(hào)土樣外,其余土樣的U與C用的機(jī)理為完全抑制作用包括競(jìng)爭(zhēng)性的和非競(jìng)爭(zhēng) to this table.均呈顯著或極顯著負(fù)相關(guān)表明土壤轉(zhuǎn)化酶活性受性的抑制作用。乙醇濃度影響顯著,乙醇對(duì)土壤轉(zhuǎn)化酶活性抑制作(4)土壤酶生態(tài)劑量(Ecological dose)中的第9期王聰穎等:乙醇對(duì)土壤轉(zhuǎn)化酶活性的影響127ED113表示土壤酶活性抑制幅度達(dá)10%時(shí)所需要加到100%時(shí),土壤轉(zhuǎn)化酶的最大表觀活性急劇降的外界污染物的濃度或劑量,通常也作為土壤受到低,與對(duì)照相比,降低了0.105mg/(g·h)。由以上輕度污染時(shí)外源污染物的臨界濃度由表3可見,不數(shù)據(jù)可知,不同體積分?jǐn)?shù)的乙醇對(duì)土壤轉(zhuǎn)化酶活性同土樣間土壤轉(zhuǎn)化酶的ED1值為29.47%~的抑制作用相差9.55倍,表明在不同底物濃度下,35.27%,最大相差僅為6.81%,表明乙醇對(duì)土壤轉(zhuǎn)乙醇對(duì)土壤轉(zhuǎn)化酶具有明顯的抑制作用?；傅淖饔檬芡寥婪柿Φ挠绊戄^小。此外,要達(dá)到輕(3)采用一元一次線性方程對(duì)酶活性(U)與乙微污染的程度,5g土樣中要含有1mL體積分?jǐn)?shù)為醇濃度(C)間關(guān)系進(jìn)行了擬合,結(jié)果見表5。由表530%~35%的乙醇溶液,這從客觀上講是不可能的可以看出,乙醇對(duì)土壤轉(zhuǎn)化酶活性具有明顯的抑制這一結(jié)果表明乙醇對(duì)土壤轉(zhuǎn)化酶的毒性不強(qiáng),即乙作用,與前面的規(guī)律是一致的,底物濃度的大小并不醇是一種污染效果不嚴(yán)重的外源污染物。影響乙醇與土壤轉(zhuǎn)化酶活性之間的關(guān)系,但其可放2.2底物濃度對(duì)乙醇與土壤轉(zhuǎn)化酶活性關(guān)系的影響大土壤轉(zhuǎn)化酶受影響的程度。由圖1可知,(1)隨蔗糖濃度增大,土壤轉(zhuǎn)化酶0.7活性持續(xù)增加。當(dāng)蔗糖濃度從0.010mol/L升高到0.100mol/L時(shí),乙醇濃度為0的土樣中土壤轉(zhuǎn)化0.6酶活性從0.280mg/(g·h)增大到0.6060.5mg/(g·h),增幅達(dá)到53.80%。0.40%;(2)在乙醇濃度相同時(shí),隨底物濃度的增加,土0.3.5%壤轉(zhuǎn)化酶活性的增加幅度減小采用模型U=A10%;0.225%BC/(1+BC)擬合底物濃度(C)與土壤酶活性0.150%(U)的關(guān)系14,結(jié)果見表4,擬合方程得到的A值就0100%是土壤酶的最大表觀酶活性。由表4可知,不同濃度00.010.0250.050.1乙醇的U和C均達(dá)到極顯著相關(guān),表明所擬合的方底物濃度(moll Substrate concentration程可較好地揭示二者之間的關(guān)系。最大表觀酶活性隨乙醇濃度增加呈先增加后降低的變化趨勢(shì),其中圖1底物濃度對(duì)乙醇與土壤轉(zhuǎn)化酶活性關(guān)系的影響乙醇體積分?jǐn)?shù)小于50%的土壤轉(zhuǎn)化酶最大表觀活 Fig. Soil invertase activity affected by different concentrations of ethanol and substrate性變幅最高為0.011mg/(gh),而當(dāng)乙醇濃度增表4土壤轉(zhuǎn)化酶活性與底物濃度關(guān)系的擬合結(jié)果 Table 4 Regression equations between soil invertase and substrate concentration乙醇體積分?jǐn)?shù)/%參數(shù) Parameter Cthanol concentrationAB00.69262.5460.99850.68862.610.998100.64868.67156.1050.998250.701.995500.70340.1130.991001.000注:自由度n-2=4=0.50.990表示顯著相關(guān);*,表示極顯著相 Note:Freedom n-2=4, ra os =0. 950,. =0. 990: remarkable correlation: .significant remarkable correlation.表5不同底物濃度下土壤轉(zhuǎn)化酶活性與乙醇濃度的擬合方程 Table 5 Regression equations between ethanol concentration and invertase activities by different substrate concentrations參數(shù)底物濃度/(mol/l) Substrate concentration Parameter0.0100.0250.0500.100A0.28030.40660.51380.6052B(10-2)0.0848460.08040.09470.10280.9350.9500.9280.900128西北農(nóng)林科技大學(xué)學(xué)報(bào)(自然科學(xué)版)第34卷2.3乙醇作用時(shí)間對(duì)土壤轉(zhuǎn)化酶活性的影響示15。反應(yīng)速度常數(shù)k反映反應(yīng)在本質(zhì)上是“快”還由圖2可知,供試土樣轉(zhuǎn)化酶活性在乙醇作用是“慢”,其值與底物濃度無關(guān)。時(shí)間為0min時(shí)最大,此后隨著作用時(shí)間的延長(zhǎng)略由表6可知,(1)與對(duì)照(乙醇體積分?jǐn)?shù)為0)相有降低其中1號(hào)和2號(hào)土樣轉(zhuǎn)化酶活性的最大降比,隨乙醇濃度的增加,除體積分?jǐn)?shù)5%乙醇處理的幅分別為16.41%和16.0%;3、4號(hào)土樣在作用時(shí)間土樣Km略有升高外,其余處理的Km值均有不同為30min時(shí)的酶活性最低,與0min相比,降幅分程度的降低,最大相差達(dá)到40%,表明乙醇的加入別為21.56%和0.92%,此后酶活性又有所增加。相導(dǎo)致土壤轉(zhuǎn)化酶與底物(蔗糖)的親合力增加,酶促關(guān)性分析結(jié)果顯示1號(hào)土樣轉(zhuǎn)化酶活性與作用時(shí)反應(yīng)更易進(jìn)行。但乙醇濃度從10%到100%時(shí),土壤間呈顯著負(fù)相關(guān)(r=-0.853,n-2=4)表明作用轉(zhuǎn)化酶酶促反應(yīng)的Km變化較小,說明乙醇濃度的時(shí)間對(duì)乙醇與土壤轉(zhuǎn)化酶活性的影響較小且變化增加對(duì)酶與底物的親合力影響較小。規(guī)律不強(qiáng)。(2)除體積分?jǐn)?shù)5%乙醇處理的土樣外,其余處理土壤轉(zhuǎn)化酶酶促反應(yīng)的Vm均小于對(duì)照。由此可no.1 -.NO.2:2.0n.3 *-NO.4知,當(dāng)乙醇體積分?jǐn)?shù)由10%增加至100%時(shí),土壤轉(zhuǎn)1.5化酶一底物復(fù)合體的解離速度逐漸降低,即乙醇對(duì)土壤轉(zhuǎn)化酶具有非競(jìng)爭(zhēng)性抑制作用161.0(3)土壤轉(zhuǎn)化酶酶促反應(yīng)的反應(yīng)速度常數(shù)k隨0.5乙醇濃度變化而變化的規(guī)律性不強(qiáng)。與對(duì)照相比,除0乙醇體積分?jǐn)?shù)為5%和100%處理的土壤轉(zhuǎn)化酶的k值等于或小于對(duì)照外,其余處理的k值均大于對(duì)100200300時(shí)間/min照,但增幅較小,最大增幅為6.77%表明在本試驗(yàn) Time的條件下,乙醇對(duì)土壤酶促反應(yīng)速度影響較小。圖2體積分?jǐn)?shù)50%乙醇作用時(shí)間對(duì)土壤(4)不同乙醇體積分?jǐn)?shù)下,土壤轉(zhuǎn)化酶的Km轉(zhuǎn)化酶活性的影響與V值呈現(xiàn)極顯著的正相關(guān)(r=0.959,n-2= Fig. Soil invertase activity affected by4),表明乙醇減弱了酶促反應(yīng)中底物與酶的親合力, 50% ethanol at different time增大了酶一底物復(fù)合物解離的速度。2.4乙醇對(duì)土壤轉(zhuǎn)化酶動(dòng)力學(xué)特征的影響(5)當(dāng)乙醇體積分?jǐn)?shù)為5%時(shí),土壤轉(zhuǎn)化酶的在米氏方程中,米氏常數(shù)Km表征酶與底物結(jié)Km和值均比對(duì)照及其余處理增大但其酶活合的牢固程度,當(dāng)Km值小時(shí),酶與底物結(jié)合牢固,性與對(duì)照相比卻減小,說明體積分?jǐn)?shù)5%的乙醇增親合力大;最大反應(yīng)速度Vmx是總酶量的量度,可表大了酶一底物復(fù)合物解離的速度,最終導(dǎo)致土壤轉(zhuǎn)征酶底物復(fù)合物分解為酶和產(chǎn)物的速率,對(duì)土壤來化酶活性降低?？梢娒竸?dòng)力學(xué)參數(shù)從多方面揭示了講其是土壤實(shí)現(xiàn)某種酶過程的潛在能力的容量指土壤酶促反應(yīng)機(jī)理等方面的差別。表61號(hào)土樣轉(zhuǎn)化酶酶促反應(yīng)的動(dòng)力學(xué)參數(shù) Table 6 Kinetic parameters of invertase of No. I soil tested Ethanol concentration (umol-L-.g-.h)Km/(mmol·l-1k/(10-5)0.46619.731.1370.608500.49222.161.1370.5670.39711.851.2140.837250.40413.251.1840.769500.43414.161.1969741000.39914.151.1000.90.986注:自由度為n-2=14,r.=0.497r0.01=0.623,示顯著相關(guān)表示極顯著相關(guān) Note Freedom n-2=14. 0. 497, a. # 0. 623; remarkable correlation; . significant remarkable.3結(jié)論活性,乙醇濃度與土壤轉(zhuǎn)化酶活性呈顯著或極顯著相關(guān),底物濃度可放大這種抑制作用;乙醇對(duì)土壤轉(zhuǎn)本試驗(yàn)結(jié)果表明,乙醇可明顯抑制土壤轉(zhuǎn)化酶化酶活性的抑制作用為完全抑制作用,包括競(jìng)爭(zhēng)性第9期王聰穎等乙醇對(duì)土壤轉(zhuǎn)化酶活性的影響129和非競(jìng)爭(zhēng)性抑制;乙醇的作用時(shí)間對(duì)土壤轉(zhuǎn)化酶活來看,乙醇是土壤中的一種輕度污染物。性有影響,但規(guī)律性不強(qiáng)所以從土壤生態(tài)毒理角度參考文獻(xiàn)] [1] Burns R G. Soil enzymes[M ] New York: Academic Press Inc. 1978.[2]周禮愷土壤酶學(xué)M]北京:科學(xué)出版社,1987.[3]關(guān)松蔭化學(xué)農(nóng)藥對(duì)土壤脲酶活性抑制作用的研究]土壤通報(bào)199223(5):232-233 [4] Sannino F,Gianfred.. Pesticide influence on soil enzymatic activities[)]. Chemosphere. 2001.451417-425. [5] Gianfreda. Sannion F. Viloante A. Pesticide effects on the activity of frec.immobilized and soil invertasc)]. Soil Biol 8. Biochem, 1995.27(9):1201-1208. [6] Baran. Jolanta E. Biclinskavet al. Enzymatic activity in an airficld soil polluted with polyeyclic aromatic hydracarbons []. Geoderma,2004(18):21-232.[7]和文祥陳會(huì)明,馮貴穎,等,汞鉻砷元素污染土壤的酶監(jiān)測(cè)研究[]環(huán)境科學(xué)報(bào)2000,203):338-3438]和文祥蔣新,朱茂旭,等甲苯對(duì)樓土酶活性影響的實(shí)驗(yàn)研究]環(huán)境科學(xué),2001.22(6):91-949]和文祥黃英鋒朱銘,等和鎘對(duì)土壤脲酶活性影響].土壤學(xué)報(bào)2002.39(3):412-420.10]關(guān)松土壤酶及其研究法[M]北京:農(nóng)業(yè)出版社,1987.11]和文祥,韋革宏,武永軍,等汞對(duì)土壤酶活性的影響中國(guó)環(huán)境科學(xué)20012(3)279-283 [12] Speir T W. Kettles H A. Parshotam A, al. Simple kinetic approach to determine the toxicity of As[] to soil biological propertics[J] Soil Biology and Biochemistry, 1999,31: 705-713. [13] Welp G. Brummer G W. Toxicity of increased amoumt of chemicals and the dose-rexponse curves for heterogeneous microbial populations in soil[]. Ecotoxicology and Environmental Safety, 1997,37: 37-44.14]和文樣朱銘,張一平汞、對(duì)土壤脲酶活性影響的研究1.尿素濃度[J]應(yīng)用生態(tài)學(xué)報(bào)2002,13(2)191-19315]和文樣,蔣新余資芬,等殺蟲雙對(duì)土壤脲酶活性特征的影響土填學(xué)報(bào)2003,40(5)50-75516]郭光基礎(chǔ)生物化學(xué)[M]北京:高等教育出版社,2001 The effect of ethanol on soil invertase activity WANG Cong-ying, HE Wen-xiang,HE Min-chao Abstract: Effect of ethanol concentration on the soil invertase activity and effect of different substrate (saccharose) concentrations on the relationship between invertase activity and ethanol were studied. The results show as follows: ethanol can remarkably inhibit soil invertase activity. The ethanol concentration and soil invertase activity are remarkably or significantly correlated in three samples. Their mechanism in- cluded direct inhibition. The ecological dose values (ED were calculated, ranging from 29. 47% to 35.27%, it shows that ethanol is a lightly toxic pollutant The substrate concentration can enlarge the influencing extent. With ethanol input, soil invertase kinetic parameters Km and values decreased, so the reaction mechanism is a kind of total inhibition between ethanol and invertase, including no-competitive and competitive. Key words: ethanol soil invertase activity kinetic characteristic; reaction mechanism