【作者】 董焱鹏；

【导师】 冯露；

【作者基本信息】 南开大学 ， 微生物学， 2012， 博士

【摘要】 一嗜热脱氮土壤芽胞杆菌NG80-2长链烷烃羟化酶LadA的体外定向进化嗜热脱氮土壤芽胞杆菌（Geobacillus thermodenitrificans）NG80-2，分离于我国大港油田，是一株能降解长链（C15-C36）烷烃的嗜热菌（最适生长温度为65℃）。在深层油藏的高温缺氧条件下，它能够利用原油为唯一碳源和能量来源，很好的生长。可用于改善原油流动性及降低重/轻质烷烃的比例。NG80-2长链烷烃代谢途径中最关键酶是第一步的长链烷烃羟化酶基因（ladA）。通过末端单加氧的方式，该酶能够催化长链烷烃（C15-C36）生成相对应的伯醇。该酶是目前能催化最长碳链的高温氧化酶，也是目前能催化长链烷烃最有用的酶之一。因此，LadA在石油污染治理中具有巨大的应用前景。本实验室前期对LadA蛋白进行了基因的克隆和表达鉴定，进行了酶学性质研究，并对其三维结构进行了x射线衍射分析。为进一步提高LadA催化长链饱和烷烃生成相对应伯醇的活性，本论文采用体外随机突变的易错PCR方法与定点饱和突变相结合的定向进化策略，以LadA野生型蛋白作为亲本，进行定向进化。通过建立合适的高通量筛选方法，最终获得了9个具有较高活性的突变酶。对这些突变酶的酶学性质进行了分析，同时对突变酶活性改变的机制与分子结构之间的联系进行了较系统的研究。建立以E. coli DH5α收集突变文库及E. coli BL21表达LadA，基于NADPH消耗的96孔板高通量筛选方法。采用易错PCR随机突变方法对ladA进行突变。通过优化实验中模版量和循环次数，将突变率控制在2-4bp/基因。建立了5个平行的突变文库，通过筛选7500个突变体，得到了3个酶活提高的突变酶（A102D,L320V和F146C/N376I）。在得到的3个突变酶4个突变位点的基础上，对这4个位点进行拆分及组合，构建了6个新酶，通过检测酶活来评估这些位点之间的相互作用。采用定点饱和突变方法对以上4个氨基酸残基及计算机预测突变“热点”114位氨基酸残基进行饱和性分析。通过测序得到了5个位点的所有氨基酸突变酶。对这些突变酶进行活性分析，得到6个高于亲本酶活性的新酶（A102E,L320A, F146Q/N376I, F146E/N376I, F146R/N376I, F146N/N376I）。对这9个突变酶（A102D, A102E, L320V，L320A, F146Q/N376I, F146E/N376I, F146R/N376I,F146N/N376I和F146C/N376I）进行基本酶学性质分析。实验结果表明，与野生型LadA相比，突变酶活性(k_cat)提高2-3.4倍，其中F146N/N376I具有最高活性；催化效率(k_cat/k_m)提高了1.9-12.7倍，其中F146E/N376I和A102E提高最多。对突变酶的最适反应温度，最适反应pH，热稳定性及底物范围等酶学性质进行了研究。表达LadA突变体的Pseudomonas fluorescens KOB2Δ1菌株比表达LadA野生型酶的KOB2Δ1菌株在以十六烷为唯一碳源和能量来源的培养基中生长快，从而在体内验证了LadA突变体的活性。对通过易错PCR技术得到的3个突变酶进行氨基酸置换。运用计算机模拟进行同源模建，进一步研究了酶活性提高与结构变化之间的关系。本研究得到的突变体在石油污染治理和原油开发等生物过程中具有很大的应用潜力。二长链脂肪酸辅酶A连接酶Facl的功能鉴定本研究对NG80-2长链烷烃降解途径中第四步的两个长链脂肪酸辅酶A连接酶（Facl1和Facl2）基因GTNG₀892和GTNG₁447进行了克隆表达和体外功能鉴定。Facl1在大肠杆菌中的表达产物分别为同二聚体，Facl2在大肠杆菌中的表达产物为非球状单体或同二聚体。Facl1和Facl2均可以催化C2到C30的脂肪酸，Facl1的最适底物是癸酸，Facl2的最适底物是棕榈酸。Facl1和Facl2最适反应温度为均为60℃，最适反应pH均为7.5，且需要ATP作为辅因子。对Facl1和Facl2的热稳定性进行了分析。同时分析了金属离子，EDTA，SDS和Triton X-100对酶活的影响。实时定量PCR显示，当NG80-2在以原油为唯一碳源的培养基中生长时，facl1和facl2的mRNA水平发生了上调。本研究对NG80-2中其余5个Facl酶也进行了克隆表达和功能鉴定。发现这5个Facl酶不能降解链长超过C14的脂肪酸，因此，可以确定Facl1和Facl2催化长链烷烃降解途径中第四步反应。这是在细菌中首次发现并鉴定的能够催化链长到至少C30的长链脂肪酸辅酶A连接酶。更多还原

【Abstract】 In vitro directed evolution of Geobacillus thermodenitrificans NG80-2LadAGeobacillus thermodenitrificans NG80-2, a described thermophilic bacterium（optimum65°C）, can degrade long-chain （C15to C36） alkanes. It is isolated from adeep-subsurface oil reservoir of Dagang oilfield, Northern China. It grows well in theanaerobic, deep subterranean oil-reservoir environment and can utilize crude oil asthe sole carbon source, thus it has the ability to enhance the fluidity of the crude oiland decrease heavy/light ratio of crude oils.LadA, a monooxygenase catalyzing the oxidation of n-alkanes to1-alkanols, isthe key enzyme for the degradation of long-chain alkanes （C15-C36） in G.thermodenitrificans NG80-2. LadA is a thermophilic monooxygenase that cancatalyze the longest chain of alkanes, to our knowledge. It is also one of the mostuseful enzymes that can utilize long chain alkanes up to now. Thus it can bepotentially used bioconvertions like pollutions and so on. During our previousresearch, the gene of LadA has been cloned, expressed and characterized. We havealso studied the enzyme characterization of LadA. What is more, the3-D structure ofLadA has also been analyzed by X-ray diffraction assays.In order to further improve its catalytic ability in hydroxylation of alkane andmake this enzyme more useful, the wild gene of LadA was aimed to further in vitrodirected evolution by error-prone PCR and saturation mutagenesis.9LadA mutantswith higher activity and higher catalytic efficiency had been found using a wellestablished high throughput screening method. Study of enzyme characteristics of themutant enzymes was researched. The molecular mechanism of the activity changeand the structure of LadA mutants was also analyzed. Directed evolution was done bycollecting the mutant libraries using E. coli DH5α,expressing LadA by E. coli BL21and screening the96-well plate based on spectroscopic assays of consumption ofNADPH. Ep-PCR was performed on the wild gene ladA. Optimized templateconcentration and cycle numbers were found and the mutant rate was controlledbetween2-4bp/gene.5similar libraries was established.7500mutants were screened. 3mutants （A102D, L320V and F146C/N376I） with higher activity were found.Site-directed mutagenesis was taken to recombine the four sites of the three mutantsand6more new enzymes were created. The6enzymes catalytic ability was analyzedto evaluate the interactions of the4sites. Saturation mutagenesis was used towardsthe four sites and another hotspot114revealed by the computer analysis. All the other19amino acids of the5sites were obtained by sequencing.6more new mutants（A102E, L320A, F146Q/N376I, F146E/N376I, F146R/N376I, F146N/N376I） wereobtained by analysis the activity of the all the mutants. Characterization of the9mutants （A102D, A102E, L320V， L320A, F146Q/N376I, F146E/N376I,F146R/N376I, F146N/N376I and F146C/N376I） was made. Comparing with thewildtype LadA, the hydroxylation activity(k_cat)of the purified LadA mutants onhexadecane was2–3.4-fold higher, with the activity of F146N/N376I being thehighest; the catalytic efficiency (k_cat/k_m) of the purified LadA mutants on hexadecanewas1.9–12.7-fold higher, with the activity of F146E/N376I and A102E being thehighest. Effects of the mutations on optimum temperature, pH and heat stability ofLadA were also investigated. A complementary study showed that Pseudomonasfluorescens KOB2Δ1strains expressing the LadA mutants grew more rapidly withhexadecane than the strain expressing wild type LadA, confirming the enhancedactivity of LadA mutants in vivo. Structural changes resulting from the mutationswere analyzed and the correlation between structural changes and enzyme activitywas discussed. The mutants generated in this study are potentially useful for thetreatment of environmental oil pollution and in other bioconversion processes.ⅡCharacterization of G. thermodenitrificans NG80-2FaclsThe functions of two long-chain fatty acid CoA ligase genes （facl） in crudeoil-degrading G. thermodenitrificans NG80-2were characterized. Facl1and Facl2encoded by GTNG₀892and GTNG₁447were expressed in Escherichia coli andpurified as His-tagged fusion proteins. Facl1was found to be a homology dimer in itsnative status while Facl2may act as a non-sphere monomer or a dimer. Both enzymesutilized a broad range of fatty acids ranging from acetic acid （C2） to melissic acid（C30）. The most preferred substrates were capric acid （C10） for Facl1and palmitic acid （C16） for Facl2, respectively. Both enzymes had an optimal temperature of60°C,an optimal pH of7.5, and required ATP as a cofactor. Thermostability of the enzymesand effects of metal ions, EDTA, SDS and Triton X-100on the enzyme activity werealso investigated. When NG80-2was cultured with crude oil rather than sucrose asthe sole carbon source, upregulation of facl1and facl2mRNA was observed by realtime RT-PCR. This is the first time that the activity of fatty acid CoA ligases towardslong-chain fatty acids up to at least C30has been demonstrated in bacteria.更多还原