| 蓝细菌脂肪醛去甲酰加氧酶的酶活分析及底物特异性研究 |
| 章晶晶
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| 导师 | 李建军
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| 2013-07
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| 学位授予单位 | 中国科学院研究生院
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| 学位授予地点 | 北京
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| 学位专业 | 生物化学与分子生物学
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| 关键词 | 生物燃料
脂肪烃
聚球藻pcc7942
脂肪醛去甲酰加氧酶
铁氧还蛋白
铁氧还蛋白还原酶
同源还原系统
酶活分析
底物特异性
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| 其他摘要 | 蓝细菌合成脂肪烃是一项利用太阳能并且无碳排放来生产先进生物燃料的过程，该过程极具工业应用价值。脂肪醛去甲酰加氧酶（aldehyde-deformylating oxygenase, ADO）催化脂肪醛转化为脂肪烃，是蓝细菌脂肪烃合成途径中的一个关键酶，属于类铁蛋白超家族。然而ADO的体外酶活非常低，针对8-18碳脂肪醛ADO没有表现出较强的底物特异性。 ADO催化的反应必须由化学还原系统（PMS/NADH）或生物还原系统（Fd/FNR/NADPH）提供电子才能发生，目前使用的生物还原系统主要是异源菠菜生物还原系统，价格非常昂贵，关于ADO同源生物还原系统的研究尚未报道。另外，控制合成特定链长的脂肪烃生物燃料意义重大。通过基因组PCR或人工合成基因方式成功得到到四种进化上亲缘关系较远蓝细菌种属的脂肪醛去甲酰加氧酶：来自点形念珠藻PCC73102的脂肪醛去甲酰加氧酶Npun_R1711、原绿球藻MIT9313的脂肪醛去甲酰加氧酶PMT1231、聚球藻PCC7942的脂肪醛去甲酰加氧酶orf1593、集胞藻PCC6803的脂肪醛去甲酰加氧酶sll0208。并成功构建相应的质粒pET28a-ADO，转化至大肠杆菌感受态细胞E.coli BL21(DE3)，经由IPTG诱导表达，实现四种不同蓝细菌来源的脂肪醛去甲酰加氧酶的异源表达。对四种脂肪醛去甲酰加氧酶催化的脂肪醛生成脂肪烃反应进行初步探索，其中orf1593催化活性最高，R1711次之，PMT1231活性很低，sll0208并未检测到活性。选择了来自聚球藻PCC7942的铁氧还蛋白Fd (SynPcc7942_1499)与铁氧还蛋白还原酶FNR (SynPcc7942_0978)作为ADO1593的还原系统。通过基因组PCR得到了铁氧还蛋白SynPcc7942_1499与铁氧还蛋白还原酶SynPcc7942_0978，构建了相应的表达载体pET28a-FNR与pET28a-Fd，在IPTG诱导条件下，在大肠杆菌 BL21(DE3)中成功实现Fd和FNR的异源表达。FNR和Fd都表现出了特征吸收峰。FNR能有效还原铁氰化钾，Km(NADPH)，kcat分别是92.5mM，226.7 s-1，不同蓝细菌种属来源的FNR对NADPH有不同亲和力。重要的是来自来自聚球藻PCC7942的Fd和FNR能有效耦合，催化细胞色素c的还原，其Km(Fd)，kcat 以及 kcat/Km值分别为15.9 mM， 85.6 s-1，and 5.4 mM-1s-1，不同蓝细菌种属来源的FNR表现出不同的细胞色素c还原酶活性。以十六碳脂肪醛为底物，分析并比较了化学还原系统PMS/NADH与生物还原系统FNR/ Fd/NADPH（来源于PCC7942或菠菜）对ADO 1593酶活的影响，结果表明：同源生物还原系统（来源于PCC7942）优于化学还原系统PMS/NADH与异源生物还原系统（来源于菠菜）；交叉实验（混合使用菠菜与聚球藻PCC7942的生物还原系统）表明ADO对Fd具有一定的选择性，并且Fd与FNR之间的电子传递对ADO活性影响很大。以庚醛为底物时，生成正己烷催化反应中，同源生物还原系统能使ADO具备更高的催化活性，比化学还原系统的kcat提高了近3.7倍。通过和中科院生物物理所合作，成功得到了orf1593的晶体结构。通过序列比对和晶体结构比对分析来自结核分枝杆菌的脂肪酸氧化酶（PDB ID code 3EE4，也属于类铁蛋白超家族）与聚球藻PCC7942的脂肪醛去甲酰加氧酶orf1593，鉴别了可能控制脂肪醛链长的氨基酸：Ala20，Ala185，Leu198。使用Stratagene公司的一步突变方法成功构建了A20Y， A185F，L198F三个单位点突变体。对每种突变体针对四种不同碳链长度的脂肪醛底物（C12-C18）进行催化活性测定，与野生型相比A20Y没有很大改变。但A185F， L198F两种突变体均使来源于聚球藻PCC7942的脂肪醛去甲酰加氧酶orf1593针对C12-C18碳链长度的脂肪醛活性降低，没有表现明显的底物选择性。正在研究对癸醛及庚醛的结果。; Biosynthesis of fatty alk(a/e)ne in cyanobacteria has been considered as a potential basis for a sunlight-driven and carbon-neutral bioprocess producing advanced solar biofuels. Aldehyde-deformylating oxygenase (ADO) is a key enzyme involved in that pathway, belonging to the ferritin-like superfamily. ADO exhibited poor catalytic activity in vitro and didn’t show strong chain-length specificity with respect to fatty aldehydes (C8-C18). Heterologous ( Fd/FNR from spinach ) or chemical ( PMS/NADH) reducing systems were generally used in in vitro ADO activity assay. Commercially available spinach Fd and FNR are extremely expensive, and a cognate electron transfer system from cyanobacteria to support ADO activity is still unknown. In addition, it is of great significance to control synthesis of fatty alkanes with the specified chain length.Four ADOs from evolutionary divergent cyanobacteria were investigated. The synthesized PMT1231 and orf1593 gene with codon optimization encoding ADOs from Prochlorococcus marinus MIT9313 and Synechococcus elongatus PCC7942 were cloned into vector pET-28a respectively. The R1711 gene obtained by genomic PCR from Nostoc punctiforme PCC73102 was also cloned into vector pET-28a. All were successfully overexpressed in E. coli BL21 (DE3) under IPTG induction. Their in vitro activities were compared, orf1593 worked best, and R1711 perfomred slightly better than PMT1231，sll0208 from PCC 6803 (whose expression construct was constructed by Aiqiu Liu in our lab) didn’t show any obvious activity. A possible endogenous reducing system including ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) was identified from the genome of Synechococcus elongates PCC7942. FNR (SynPcc7942_0978), and Fd (SynPcc7942_1499) from PCC7942 were cloned into vector pET-28a, overexpressed, purified, and characterized. FNR showed a characteristic FAD absorption spectrum and the UV/vis spectrum of Fd clearly displayed three peaks characteristic of the presence of the [2Fe-2S] cluster. FNR from PCC7942 was active against potassium ferricyanide with Km(NADPH) 92.5 mM and kcat 226.7 s-1. Importantly, Fd and FNR from PCC7942 were functionally coupled in cytochrome c reduction with Km 15.9 mM and kcat 85.6 s-1. FNRs from evolutionarily divergent cyanobacteria exhibited different binding affinity for NADPH and catalytic efficiency towards ferricyanide reduction, and different kinetic behaviour against Fd-mediated reduction of cytochrome c.ADO 1593 was in vitro reconstituted with Fd and FNR from PCC7942, and tested for bioconversion of n-hexadecanal. ADO activity was successfully supported by the endogenous Fd/FNR system, which worked more effective than the heterologous Fd/FNR and chemical ones. The hybrid experiments of the surrogate and cognate biological reducing systems demonstrated that ADO is selective against Fd and interaction between Fd and FNR is very important for the efficient electron transfer and ADO activity. Importantly, the kcat value of ADO 1593 using the homologous Fd/FNR electron transfer system is 3.7-fold higher than that for the chemical one when using heptanal as substrate. A crystal structure of the PCC7942_orf1593 was solved by cooperating with a group from Institute of Biophysics, Chinese Academy of Science. Based on the protein sequence and crystal structure ( PDB ID code 3EE4, belonging to the ferritin-like superfamily) of fatty acid oxidase from Mycobacterium tuberculosis and ADO 1593 from Synechococcus elongatus PCC7942, Ala20，Ala185，Leu198 of 1593 which could affect chain-length selectivity of fatty aldehydes were identified. Mutants A20Y, A185F and L198F were constructed by site-directed mutagenesis. Their catalytic activities towards fatty aldehydes (C12-C18) with different chain length were investigated and compared. Compared with WT 1593, the activity didn’t change a lot , whereas the other two muants showed lower activity. Their selectivities against decanal and heptanal are being investigated. |
| 作者部门 | 生物代谢工程 |
| 学科领域 | 生物代谢工程
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| 公开日期 | 2013-07-13
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| 学位类型 | 硕士
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| 语种 | 中文
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| 文献类型 | 学位论文
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| 条目标识符 | http://ir.qibebt.ac.cn/handle/337004/1520
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| 专题 | 微生物代谢工程研究组
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推荐引用方式
GB/T 7714 |
章晶晶. 蓝细菌脂肪醛去甲酰加氧酶的酶活分析及底物特异性研究[D]. 北京. 中国科学院研究生院,2013.
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