微生物降解烷烃的研究

1、Degradation of alkanes by bacteria岳靓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 综述综述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 介绍介绍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 介绍介绍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-烷烃降解细菌专一菌种与非专一菌种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-烷烃降解细菌专一菌种与非专一菌种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-烷烃降解细菌烷烃降解细菌专一菌种与非专一菌种专一菌种与非专一菌种不同的细菌对烷烃的降解情况也各不相同。不优先利用烷烃作底物的菌高度专业化的菌典型的烃降解菌（ hydrocarbonoclastic bacteria ）Alkane-degrading bacteria: specialized and non-specialized species-烷烃降解细菌烷烃降解细菌专一菌

16、种与非专一菌种专一菌种与非专一菌种-食烷菌（Alcanivorax borkumensis,）降解链烃，支链烃，但不降解芳香烃、糖类、氨基酸、脂肪酶和其他常见的碳源。食烷菌存在于未污染的海水中，他们可能依靠藻类及其他海洋生物产生的烷烃生存，他们数量不多，但相对稳定。与之功能类似的菌还有深海弯曲菌属（genera Thalassolituus）、嗜油菌属（genera Thalassolituus）、油螺旋菌属（Oleispira）Uptake of n-alkanes-链烃的降解烷烃在水中的溶解度小，且分子量越大，溶解性越小。这阻碍了微生物对其的降解。对于烷烃如何进入细胞还未有定论。低分子量的

17、烷烃在水中具有足够的溶解性，这就可以保证足量的物质转移到细胞中。对于中链及长链的烷烃，微生物要么通过粘附在烃类油滴上，要么通过表面活性物质促进吸收。表面活性剂可以提高烃类在液体的溶解度，但对于土壤或其它状态下是无效的。Aerobic degradation of alkanes-烷烃的有氧降解烷烃的有氧降解是让O2作为电子受体。用来激活烷烃的酶是一种单氧酶，某些好氧菌种生产这种酶，它可以克服烃类化合物的低化学活性。Aerobic degradation of alkanes-烷烃的有氧降解-甲烷氧化甲烷氧化成甲醇，再到甲醛，最后到甲酸，这就是一个典型的氧化反应Aerobic degradati

18、on of alkanes-烷烃的有氧降解-多碳烷烃氧化本文介绍的链烷烃的降解方式有两种：单末端氧化、和次末端氧化。单末端氧化是在加氧酶的作用下，氧直接结合到碳链末端的碳上，氧结合到碳链末端形成伯醇，伯醇再依次氧化成对应的醛和脂肪酸；次末端氧化指从烷烃末端第二个碳开始氧化，形成仲醇，然后再依次氧化成酮和脂，脂被水解为伯醇和乙酸后进一步分解。Aerobic degradation of alkanes-烷烃的有氧降解Methane monooxygenases and related-甲烷单加氧酶及相关的烷烃羟化酶 甲烷单加氧酶会产生一种膜结合甲烷单加氧酶微粒particulate methan

19、e monooxygenase (pMMO)，它们其中有一小部分也包含一种可溶性甲烷单加氧酶soluble methane monooxygenase (sMMO)。如果一支菌株同时含有pMMO和 sMMO ，sMMO仅仅在可用铜离子浓度很低的情况下才表达。sMMO羟化酶还原酶调控蛋白pMMO PmoA PmoB PmoCAerobic degradation of alkanes-烷烃的有氧降解Methane monooxygenases and related-甲烷单加氧酶及相关的烷烃羟化酶 一些细菌不能利用甲烷生长，但可利用C2-C4烷烃。eg.butanovora 假单胞菌（Pseud

20、omonas butanovora），可对C2-C4烷烃进行连续的一系列的末端氧化。这个过程最先需要的酶是丁烷单加氧酶butane monooxygenase (BMO)，这是一种与sMMO相似的血红素铁单加氧酶，而合成这种酶需要一种伴侣蛋白BmoG。戈登式菌属Gordonia sp. TY-5可利用丙烷作为碳源 它可利用一种底物利用相对单一的sMMO相似的酶次末端氧化丙烷。相似的菌还有Mycobacterium sp. TY-6 and Pseudono- cardia sp. TY-7。Aerobic degradation of alkanes-烷烃的有氧降解The AlkB famil

21、y of alkane hydroxylases-膜结合烷烃羟化酶族最优特点的烷烃降解途径是恶臭假单胞菌Gpo1（之前叫做食油假单胞菌Gpo1）被编码的OCT质粒。第一个酶是AlkB，在末端位置羟化烷烃。AlkB需要两个可溶性电子转移蛋白叫红氧还蛋白（AlkG）和红素氧还蛋白还原酶（AlkT）。红素氧还蛋白还原酶，通过它的辅因子FAD，将电子从NADH转移到红素氧还蛋白，交替向AlkB转移电子。虽然AlkB没有晶体结构，但是它具有六个跨膜片段以及作用于细胞质的催化基团。活性部位包括4个含有组氨酸的序列并保留有其它烷烃单氧酶的基元，并且螯合了两个铁原子。双铁原子族通过底物自由基使烷烃有氧激活。其

22、中一个氧原子被转移到烷烃甲基末端，变成羟基，另一个氧原子被血红氧还蛋白通过电子转移转化成H2O。氧化是具有区域选择性和立体专一性的。Aerobic degradation of alkanes-烷烃的有氧降解The AlkB family of alkane hydroxylases-膜结合烷烃羟化酶族现有超过60种AlkB酶的同系物，并显示出很高的序列多样性。蛋白红素氧还蛋白 （rubredoxin）传递给AlkB酶的活性位点是一个体积很小的铁硫氧化还原蛋白（ redox-active iron-sulfur protein ）。 P. putida GPo1 AlkG含有两个红素氧还蛋白类

23、型AlkG1 and AlkG2，而其它微生物的红素氧还蛋白只有其中一种。AlkG1功能未知， AlkG2可传递电子。Aerobic degradation of alkanes-烷烃的有氧降解Cytochrome P450 alkane hydroxylases-细胞色素P450烷烃羟化酶细胞色素P450是羟化大量化合物的血红素蛋白，它们在生物圈中无所不在，根据序列相似性被分为100多个族。一些可降解C5-C10烷烃的细菌含不同的P450。eg. 不动杆菌属Acinetobacter sp. EB104 CYP153A1 相似的酶还存在于分支杆菌属mycobacteria,红球菌属rhodo

24、cocci 及变形菌属proteobacteria。Aerobic degradation of alkanes-烷烃的有氧降解Alkane hydroxylases for long-chain n-alkanes -长直链烷烃的降解一些细菌能够降解C20的长直链烃，这些细菌常常带有一些烷烃羟化酶。其对于C10-C20的降解常与AlkB和P450相关，但当C20时作用酶则与上二者完全无关。eg. Acinetobacter sp. M1, 可以以C13C44 烷烃为底物生长, 包含一个可溶性 Cu2+的烷烃羟化酶，其 对C10C30 烷烃显示活性，它可能是一种双加氧酶可将氢过氧化物氧化产生相

25、应的醛类。Aerobic degradation of alkanes-烷烃的有氧降解Metabolism of the alcohols and aldehydes derived from the oxidation of alkanes -烷烃氧化产生的醇醛的分解代谢1.末端氧化产物 烷烃末端氧化产生脂肪酸乙醇脱氢酶（ADH）醛不同的乙醇脱氢酶降解不同的烷烃2.次末端氧化产物烷烃末端氧化产生仲醇ADH酮Aerobic degradation of alkanes-烷烃的有氧降解Metabolism of the alcohols and aldehydes derived from th

26、e oxidation of alkanes -烷烃氧化产生的醇醛的分解代谢一些菌种包含几种不同的ADH，这样可以用来分解不同的醇类。eg1. 针对伯醇和仲醇，假单胞菌 butanovora最 少用四种不同的ADH。eg2.醋酸钙不动杆菌HO1-N 则至少有两种ADH 一种对正葵醇表现出活性，另一种则对十四醇表现活性。Aerobic degradation of alkanes-烷烃的有氧降解Degradation of branched-chain alkanes -支链烷烃的降解支链烷烃较之直链烷烃是难降解的，但仍有几种菌可以降解异辛烷和姥鲛烷。eg. Alcanivorax sp.可降解

27、姥鲛烷和植烷，所以它在降解含油海洋水体方面有较广阔的应用。Anaerobic degradation of alkanes-烷烃的厌氧降解烷烃的厌氧降解是烃类在环境中的循环中重要的一步。厌氧降解指在严格厌氧环境下，细菌降解烷烃不依靠氧气，而是将硝酸盐和硫酸盐作为电子受体。厌氧降解的过程要比有氧降解过程慢，且细菌对可利用的烷烃的要求更加严苛。（eg. strain BuS5只能降解丙烷和丁烷,etc.）氧化代谢氧化的一个长链脂肪酸通过连续周期的反应在每一步的脂肪酸是缩短形成含两个原子碎片移除乙酰辅酶A。Organization of alkane-degradation genes-烷烃降解基因

28、不同的烷烃降解菌对烷烃的降解有很大差异。P. putida GPo1 OCT质粒编码的烷烃降解基因集中于两个操纵子上，对许多细菌来说，这种降解途径显然是通过水平转移的。如果一个细菌中有多个烷烃羟化酶，那么通常，这些基因位于细菌染色体的不同位置上。此外，这些或远或近的基因的表达上会出现重叠。Regulation of alkane-degradation pathways-烷烃降解途径的调控参与烷烃内部氧化的基因表达的控制是十分严密的。调控剂的专一性表现在只有当确定的烷烃出现时，基因才会表达。已知诱导烷烃降解基因的不同的调控蛋白与对于不同家族的烷烃具有专一性。eg. LuxR/MalT, theAraC/XylS, the GntR。有证据证明长链烷烃和长链烯烃作为效应剂。因为烷烃是非极性分子，所以其最可能聚集到细胞质膜上，转录调节器是胞浆蛋白。所以问题在调控蛋白如何与烷烃反应。 A. borkumensis AlkS转录调节器可以激活AlkB1和与烷烃相关的下游基因编码。在一个有关蛋白质组成的研究中调控蛋白展示出其与膜部分的关联性大于与细胞质的关联性。尽管AlkS没有表现出预想的膜蛋白性质，但它也许对细胞质内侧膜表现出亲和力，在这里，烷烃易作为效应体。绑定了烷烃后， AlkS应当移动并找到DNA上的绑定位点。 Regulation o