微生物降解烷烴的研究

1、Degradation of alkanes by bacteria岳靚142083002012Degradation of alkanes by bacteria-Summary-Introduction-Alkane-degrading bacteria: specialized and non-specialized species-Uptake of n-alkanes-Aerobic degradation of alkanes-Anaerobic degradation of alkanes-Organization of alkane-degradation genes-Regu

2、lation of alkane-degradation pathways-Converting excess carbon into storage materials-Concluding remarksSummary 綜述綜述Pollution of soil and water environments by crude oilhas been, and is still today, an important problem.Crude oil is a complex mixture of thousands of compounds.Among them, alkanes con

3、stitute the majorfraction. Alkanes are saturated hydrocarbons of differentsizes and structures. Although they are chemicallyvery inert, most of them can be efficientlydegraded by several microorganisms. This reviewsummarizes current knowledge on how microorganismsdegrade alkanes, focusing on the bio

4、chemicalpathways used and on how the expression ofpathway genes is regulated and integrated within cellphysiology.Introduction 介紹介紹Alkanes are saturated hydrocarbons, formed exclusively by carbon and hydrogen atoms. They can be linear (n-alkanes), cyclic (cyclo-alkanes) or branched (iso- alkanes). T

5、hose having between one and four carbon atoms (methane to butane) are gaseous at ambient temperature. Larger molecules are liquid or solid.Alkanes can constitute up to 50% of crude oil, depending on the oil source, but are also produced by many living organisms such as plants, green algae, bacteria

6、or animals. This probably explains why alkanes are present at low concentrations in most soil and water environments. As alkanes are apolar molecules that are chemically very inert (Lab- ingerandBercaw,2002),their metabolism by microorganisms poses challenges related to their low water solubility, t

7、heir tendency to accumulate in cell membranes, and the energy needed to activate the molecule.However,several microorganisms, both aerobic and anaerobic, can use.Introduction 介紹介紹diverse alkanes as a source of carbon and energy. Several reviews have covered different aspects of the physiology, enzym

8、es and pathways responsible for the degradation of alkanes (Watkinson and Morgan, 1990; Ashraf et al., 1994; van Beilen et al., 2003; van Hamme et al., 2003; Coon, 2005; van Beilen and Funhoff, 2007; Wentzel et al., 2007), so that this review is devoted to stress recent findings and how the expressi

9、on of the alkane-degradation genes is regulated.Alkane-degrading bacteria: specialized and non-specialized species-烷烴降解細菌專一菌種與非專一菌種Many microorganisms (bacteria, filamentous fungi and yeasts) can degrade alkanes, using them as the carbon source. A typical soil, sand or ocean sediment contains signif

10、icant amounts of hydrocarbon-degrading microorganisms, and their numbers increase considerably in oil-polluted sites. Various alkane degraders are bacteria that have a very versatile metabolism, so that they can use as carbon source many other compounds in addition to alkanes. Most frequently, alkan

11、es are not preferred growth substrates for these bacteria, which will rather utilize other compounds before turning to alkanes. On the other hand, some bacterial species are highly specialized in degrading hydro- carbons. They are called hydrocarbonoclastic bacteria and play a key role in the remova

12、l of hydrocarbons from polluted environments.Alkane-degrading bacteria: specialized and non-specialized species-烷烴降解細菌專一菌種與非專一菌種Many microorganisms (bacteria, filamentous fungi and yeasts) can degrade alkanes, using them as the carbon source. A typical soil, sand or ocean sediment contains significa

13、nt amounts of hydrocarbon-degrading microorganisms, and their numbers increase considerably in oil-polluted sites. Various alkane degraders are bacteria that have a very versatile metabolism, so that they can use as carbon source many other compounds in addition to alkanes. Most frequently, alkanes

14、are not preferred growth substrates for these bacteria, which will rather utilize other compounds before turning to alkanes. On the other hand, some bacterial species are highly specialized in degrading hydro- carbons. They are called hydrocarbonoclastic bacteria and play a key role in the removal o

15、f hydrocarbons from polluted environments.Alkane-degrading bacteria: specialized and non-specialized species-烷烴降解細菌烷烴降解細菌專一菌種與非專一菌種專一菌種與非專一菌種不同的細菌對烷烴的降解情況也各不相同。不優(yōu)先利用烷烴作底物的菌高度專業(yè)化的菌典型的烴降解菌（ hydrocarbonoclastic bacteria ）Alkane-degrading bacteria: specialized and non-specialized species-烷烴降解細菌烷烴降解細菌專一菌

16、種與非專一菌種專一菌種與非專一菌種-食烷菌（Alcanivorax borkumensis,）降解鏈烴，支鏈烴，但不降解芳香烴、糖類、氨基酸、脂肪酶和其他常見的碳源。食烷菌存在于未污染的海水中，他們可能依靠藻類及其他海洋生物產(chǎn)生的烷烴生存，他們數(shù)量不多，但相對穩(wěn)定。與之功能類似的菌還有深海彎曲菌屬（genera Thalassolituus）、嗜油菌屬（genera Thalassolituus）、油螺旋菌屬（Oleispira）Uptake of n-alkanes-鏈烴的降解烷烴在水中的溶解度小，且分子量越大，溶解性越小。這阻礙了微生物對其的降解。對于烷烴如何進入細胞還未有定論。低分子量的

17、烷烴在水中具有足夠的溶解性，這就可以保證足量的物質(zhì)轉(zhuǎn)移到細胞中。對于中鏈及長鏈的烷烴，微生物要么通過粘附在烴類油滴上，要么通過表面活性物質(zhì)促進吸收。表面活性劑可以提高烴類在液體的溶解度，但對于土壤或其它狀態(tài)下是無效的。Aerobic degradation of alkanes-烷烴的有氧降解烷烴的有氧降解是讓O2作為電子受體。用來激活烷烴的酶是一種單氧酶，某些好氧菌種生產(chǎn)這種酶，它可以克服烴類化合物的低化學活性。Aerobic degradation of alkanes-烷烴的有氧降解-甲烷氧化甲烷氧化成甲醇，再到甲醛，最后到甲酸，這就是一個典型的氧化反應Aerobic degradati

18、on of alkanes-烷烴的有氧降解-多碳烷烴氧化本文介紹的鏈烷烴的降解方式有兩種：單末端氧化、和次末端氧化。單末端氧化是在加氧酶的作用下，氧直接結合到碳鏈末端的碳上，氧結合到碳鏈末端形成伯醇，伯醇再依次氧化成對應的醛和脂肪酸；次末端氧化指從烷烴末端第二個碳開始氧化，形成仲醇，然后再依次氧化成酮和脂，脂被水解為伯醇和乙酸后進一步分解。Aerobic degradation of alkanes-烷烴的有氧降解Methane monooxygenases and related-甲烷單加氧酶及相關的烷烴羥化酶 甲烷單加氧酶會產(chǎn)生一種膜結合甲烷單加氧酶微粒particulate methan

19、e monooxygenase (pMMO)，它們其中有一小部分也包含一種可溶性甲烷單加氧酶soluble methane monooxygenase (sMMO)。如果一支菌株同時含有pMMO和 sMMO ，sMMO僅僅在可用銅離子濃度很低的情況下才表達。sMMO羥化酶還原酶調(diào)控蛋白pMMO PmoA PmoB PmoCAerobic degradation of alkanes-烷烴的有氧降解Methane monooxygenases and related-甲烷單加氧酶及相關的烷烴羥化酶 一些細菌不能利用甲烷生長，但可利用C2-C4烷烴。eg.butanovora 假單胞菌（Pseud

20、omonas butanovora），可對C2-C4烷烴進行連續(xù)的一系列的末端氧化。這個過程最先需要的酶是丁烷單加氧酶butane monooxygenase (BMO)，這是一種與sMMO相似的血紅素鐵單加氧酶，而合成這種酶需要一種伴侶蛋白BmoG。戈登式菌屬Gordonia sp. TY-5可利用丙烷作為碳源 它可利用一種底物利用相對單一的sMMO相似的酶次末端氧化丙烷。相似的菌還有Mycobacterium sp. TY-6 and Pseudono- cardia sp. TY-7。Aerobic degradation of alkanes-烷烴的有氧降解The AlkB famil

21、y of alkane hydroxylases-膜結合烷烴羥化酶族最優(yōu)特點的烷烴降解途徑是惡臭假單胞菌Gpo1（之前叫做食油假單胞菌Gpo1）被編碼的OCT質(zhì)粒。第一個酶是AlkB，在末端位置羥化烷烴。AlkB需要兩個可溶性電子轉(zhuǎn)移蛋白叫紅氧還蛋白（AlkG）和紅素氧還蛋白還原酶（AlkT）。紅素氧還蛋白還原酶，通過它的輔因子FAD，將電子從NADH轉(zhuǎn)移到紅素氧還蛋白，交替向AlkB轉(zhuǎn)移電子。雖然AlkB沒有晶體結構，但是它具有六個跨膜片段以及作用于細胞質(zhì)的催化基團?；钚圆课话?個含有組氨酸的序列并保留有其它烷烴單氧酶的基元，并且螯合了兩個鐵原子。雙鐵原子族通過底物自由基使烷烴有氧激活。其

22、中一個氧原子被轉(zhuǎn)移到烷烴甲基末端，變成羥基，另一個氧原子被血紅氧還蛋白通過電子轉(zhuǎn)移轉(zhuǎn)化成H2O。氧化是具有區(qū)域選擇性和立體專一性的。Aerobic degradation of alkanes-烷烴的有氧降解The AlkB family of alkane hydroxylases-膜結合烷烴羥化酶族現(xiàn)有超過60種AlkB酶的同系物，并顯示出很高的序列多樣性。蛋白紅素氧還蛋白 （rubredoxin）傳遞給AlkB酶的活性位點是一個體積很小的鐵硫氧化還原蛋白（ redox-active iron-sulfur protein ）。 P. putida GPo1 AlkG含有兩個紅素氧還蛋白類

23、型AlkG1 and AlkG2，而其它微生物的紅素氧還蛋白只有其中一種。AlkG1功能未知， AlkG2可傳遞電子。Aerobic degradation of alkanes-烷烴的有氧降解Cytochrome P450 alkane hydroxylases-細胞色素P450烷烴羥化酶細胞色素P450是羥化大量化合物的血紅素蛋白，它們在生物圈中無所不在，根據(jù)序列相似性被分為100多個族。一些可降解C5-C10烷烴的細菌含不同的P450。eg. 不動桿菌屬Acinetobacter sp. EB104 CYP153A1 相似的酶還存在于分支桿菌屬mycobacteria,紅球菌屬rhodo

24、cocci 及變形菌屬proteobacteria。Aerobic degradation of alkanes-烷烴的有氧降解Alkane hydroxylases for long-chain n-alkanes -長直鏈烷烴的降解一些細菌能夠降解C20的長直鏈烴，這些細菌常常帶有一些烷烴羥化酶。其對于C10-C20的降解常與AlkB和P450相關，但當C20時作用酶則與上二者完全無關。eg. Acinetobacter sp. M1, 可以以C13C44 烷烴為底物生長, 包含一個可溶性 Cu2+的烷烴羥化酶，其 對C10C30 烷烴顯示活性，它可能是一種雙加氧酶可將氫過氧化物氧化產(chǎn)生相

25、應的醛類。Aerobic degradation of alkanes-烷烴的有氧降解Metabolism of the alcohols and aldehydes derived from the oxidation of alkanes -烷烴氧化產(chǎn)生的醇醛的分解代謝1.末端氧化產(chǎn)物 烷烴末端氧化產(chǎn)生脂肪酸乙醇脫氫酶（ADH）醛不同的乙醇脫氫酶降解不同的烷烴2.次末端氧化產(chǎn)物烷烴末端氧化產(chǎn)生仲醇ADH酮Aerobic degradation of alkanes-烷烴的有氧降解Metabolism of the alcohols and aldehydes derived from th

26、e oxidation of alkanes -烷烴氧化產(chǎn)生的醇醛的分解代謝一些菌種包含幾種不同的ADH，這樣可以用來分解不同的醇類。eg1. 針對伯醇和仲醇，假單胞菌 butanovora最 少用四種不同的ADH。eg2.醋酸鈣不動桿菌HO1-N 則至少有兩種ADH 一種對正葵醇表現(xiàn)出活性，另一種則對十四醇表現(xiàn)活性。Aerobic degradation of alkanes-烷烴的有氧降解Degradation of branched-chain alkanes -支鏈烷烴的降解支鏈烷烴較之直鏈烷烴是難降解的，但仍有幾種菌可以降解異辛烷和姥鮫烷。eg. Alcanivorax sp.可降解

27、姥鮫烷和植烷，所以它在降解含油海洋水體方面有較廣闊的應用。Anaerobic degradation of alkanes-烷烴的厭氧降解烷烴的厭氧降解是烴類在環(huán)境中的循環(huán)中重要的一步。厭氧降解指在嚴格厭氧環(huán)境下，細菌降解烷烴不依靠氧氣，而是將硝酸鹽和硫酸鹽作為電子受體。厭氧降解的過程要比有氧降解過程慢，且細菌對可利用的烷烴的要求更加嚴苛。（eg. strain BuS5只能降解丙烷和丁烷,etc.）氧化代謝氧化的一個長鏈脂肪酸通過連續(xù)周期的反應在每一步的脂肪酸是縮短形成含兩個原子碎片移除乙酰輔酶A。Organization of alkane-degradation genes-烷烴降解基因

28、不同的烷烴降解菌對烷烴的降解有很大差異。P. putida GPo1 OCT質(zhì)粒編碼的烷烴降解基因集中于兩個操縱子上，對許多細菌來說，這種降解途徑顯然是通過水平轉(zhuǎn)移的。如果一個細菌中有多個烷烴羥化酶，那么通常，這些基因位于細菌染色體的不同位置上。此外，這些或遠或近的基因的表達上會出現(xiàn)重疊。Regulation of alkane-degradation pathways-烷烴降解途徑的調(diào)控參與烷烴內(nèi)部氧化的基因表達的控制是十分嚴密的。調(diào)控劑的專一性表現(xiàn)在只有當確定的烷烴出現(xiàn)時，基因才會表達。已知誘導烷烴降解基因的不同的調(diào)控蛋白與對于不同家族的烷烴具有專一性。eg. LuxR/MalT, theAraC/XylS, the GntR。有證據(jù)證明長鏈烷烴和長鏈烯烴作為效應劑。因為烷烴是非極性分子，所以其最可能聚集到細胞質(zhì)膜上，轉(zhuǎn)錄調(diào)節(jié)器是胞漿蛋白。所以問題在調(diào)控蛋白如何與烷烴反應。 A. borkumensis AlkS轉(zhuǎn)錄調(diào)節(jié)器可以激活AlkB1和與烷烴相關的下游基因編碼。在一個有關蛋白質(zhì)組成的研究中調(diào)控蛋白展示出其與膜部分的關聯(lián)性大于與細胞質(zhì)的關聯(lián)性。盡管AlkS沒有表現(xiàn)出預想的膜蛋白性質(zhì)，但它也許對細胞質(zhì)內(nèi)側膜表現(xiàn)出親和力，在這里，烷烴易作為效應體。綁定了烷烴后， AlkS應當移動并找到DNA上的綁定位點。 Regulation o