节杆菌黄嘌呤氧化酶生物合成调控与酶稳定性研究

发布时间：2018-09-07 18:43

【摘要】：黄嘌呤氧化酶（Xanthine oxidase，EC1.17.3.2，XOD）属于黄素蛋白氧化酶类中较为复杂的多亚基蛋白，其底物催化机理复杂，除需辅因子FAD，还需钼蝶呤及铁硫簇辅因子。这三种辅因子以严格的比例及特定的顺序排布于XOD结构内部，联合最终电子受体（分子氧），，共同催化嘌呤类物质的降解。相比于黄嘌呤脱氢酶（Xanthinedehydrogenase，EC1.17.1.4，XDH），XOD在医学诊断、食品检测、工业催化及环境保护中的应用价值更为广泛。本论文以一株拥有自主知识产权的XOD产生株—节杆菌（Arthrobacter）M3为研究对象，探讨发酵合成XOD的调控技术；合成筛选含特异性配体的亲和介质，创建简易亲和纯化XOD的方法，并分析酶热不稳定机理；探寻可有效提高XOD稳定性的技术；研究次黄嘌呤/黄嘌呤降解代谢产物与XOD合成的关系，利用等离子体诱变，半定向筛选代谢产物抑制得到衰减的突变株。主要研究结果如下：考察次黄嘌呤（诱导物）及辅因子添加对XOD合成的影响，发现，次黄嘌呤最佳诱导浓度为3.6g L-1，而添加辅因子前体核黄素（0.30mg L-1）及硫胺素（6.0mg L-1）可分别使XOD平均产率提高17.0%和16.3%。根据不同pH值控制下Arthrobacter M3的发酵过程曲线和动力学参数变化，提出了分段式pH调控技术，即在发酵前期以初始pH8.6进行自然发酵，待菌体密度至2.0g L-1时，控制发酵液pH值为7.6。该分段式pH调控技术的应用，使XOD平均产率（1229.7U g-1）比单独使用恒pH7.6发酵和自然pH发酵（初始pH8.6）时分别提高了86.3%和89.4%；酶活水平（7415.3U L-1）分别提高了75.0%和91.0%。以Logistic方程和Luedeking-Piret方程描述了分段式pH发酵过程中菌体生长、XOD积累以及基质（残糖）消耗的模型，模型相关系数（R2）均大于0.97。分别以鸟嘌呤（黄嘌呤结构类似物）及核黄素（FAD前体）为配体，合成了亲和介质（琼脂糖为载体）。吸附分析表明，琼脂糖-鸟嘌呤亲和介质对XOD的吸附能力较佳（2.0mg g-1介质），并利用液质联用技术证实了鸟嘌呤配体与琼脂糖载体的成功偶联。采用硫酸铵盐析、琼脂糖-鸟嘌呤亲和层析及DEAE-Sepharose CL-4B离子交换层析共3步法，简易地纯化了Arthrobacter M3XOD，比酶活为1033.2U mg-1，纯化倍数为120.1，回收率为36.1%。研究Arthrobacter M3XOD的酶学性质则表明：XOD为含有两个亚基（100kDa和35kDa）的异质二聚体蛋白，相对分子质量135kDa，与Arthrobacter sp. FB24XDH的匹配度较高（肽质量指纹图谱分析）；最适反应温度为37℃，最适反应pH为7.5，且对不同金属离子（2.0mmol L-1）的耐受性具有差异，对黄嘌呤的动力学常数Km为0.67mmol L-1。对50℃保温不同时间的XOD酶液进行分析，发现XOD热不稳定机理主要为疏水基团暴露引起的蛋白集聚。以XOD的热不稳定机理为指导，针对性的添加了海藻糖和甜菜碱等保护剂。结果表明，添加1.0mol L-1海藻糖可使XOD半衰期（50℃）延长至6.9h（对照组仅为0.84h），凝胶过滤色谱分析表明海藻糖的加入有效抑制了蛋白集聚。分别选用阴离子交换树脂（201×4、D201及D354）离子吸附固定化、聚丙烯酰胺及海藻酸钠物理包埋固定化、疏水性载体（D840）及亲水性载体（含不同间隔臂长度的琼脂糖）共价偶联固定化处理XOD，不同程度提高了酶热稳定性。其中，以海藻酸钠物理包埋的固定化酶酶活回收率较高（17.3%）；50℃保温2h，以琼脂糖-乙二胺载体共价偶联的固定化酶相对酶活保留率较高（90.9%）。将琼脂糖-乙二胺载体固定化的酶与游离酶比较，固定化酶于不同pH和温度下的耐受性均提高，半衰期延长至5.5h（50℃），重复使用8次后，相对酶活仍保留41.2%。研究次黄嘌呤/黄嘌呤降解代谢途径中关键代谢产物（自由基、尿酸、尿素及铵根离子）与XOD合成的关系，发现，在XOD发酵合成过程中，嘌呤代谢含氮终产物—铵根离子是抑制XOD合成的关键因子；积累的代谢中间产物尿素对XOD合成基本无影响，且积累浓度（1.10g L-1）远低于其抑制XOD合成的浓度（≥15.0g L-1）。为减弱次黄嘌呤/黄嘌呤降解代谢途径中尿素向铵根离子的降解，采用等离子体诱变，经三级筛选平板初筛，半定向选育了一株低产尿素降解酶的突变株Arthrobacter M605，与出发菌株Arthrobacter M3相比，其尿素降解酶平均产率降低了49.3%。补料分批发酵条件下，突变株Arthrobacter M605的XOD平均产率及酶活水平提高至1168.5U g-1和12970.6U L-1，分别比出发菌株Arthrobacter M3提高了38.6%和42.5%。
[Abstract]:Xanthine oxidase (EC 1.17.3.2, XOD) is a kind of complex multi-subunit protein in flavin protein oxidase. Its substrate catalytic mechanism is complex, besides the cofactor FAD, it also needs molybdenum pterine and iron sulfur cluster cofactor. These three cofactors are arranged in the structure of XOD in strict proportion and specific order, and combine with the final electron acceptance. Compared with Xanthine dehydrogenase (EC 1.17.1.4, XDH), XOD is more widely used in medical diagnosis, food detection, industrial catalysis and environmental protection.
In this paper, Arthrobacter M3, a XOD producing strain with independent intellectual property rights, was used as the research object to explore the regulation technology of XOD synthesis by fermentation; to synthesize and screen affinity media containing specific ligands; to create a simple affinity purification method of XOD; to analyze the mechanism of enzyme thermal instability; to explore the technology that can effectively improve the stability of XOD. To study the relationship between hypoxanthine/xanthine degradation metabolites and XOD synthesis, a semidirectional screening of attenuated mutants by plasma mutagenesis was carried out.
The effects of hypoxanthine (inducer) and cofactor addition on the synthesis of XOD were investigated. It was found that the optimal induction concentration of hypoxanthine was 3.6 g L-1, and the addition of cofactor precursor riboflavin (0.30 mg L-1) and thiamine (6.0 mg L-1) increased the average yield of XOD by 17.0% and 16.3% respectively. With the change of kinetic parameters, a segmented pH control technique was proposed, i.e. natural fermentation at initial pH 8.6 in the early stage of fermentation and pH 7.6 in the fermentation broth when cell density was 2.0 g L-1. The application of the segmented pH control technique made the average yield of XOD (1229.7 U g-1) higher than that of fermentation at constant pH 7.6 and natural pH (initial pH 8.6) respectively. Enzyme activity (7415.3U L-1) was increased by 75.0% and 91.0% respectively. The models of cell growth, XOD accumulation and substrate (residual sugar) consumption were described by Logistic equation and Luedeking-Piret equation. The model correlation coefficient (R2) was greater than 0.97.
The affinity medium (agarose as carrier) was synthesized by using guanine (xanthine structural analogue) and riboflavin (FAD precursor) as ligands. The adsorption analysis showed that the affinity medium of agarose-guanine had better adsorption ability for XOD (2.0 mg g-1 medium). The coupling of guanine ligand with agarose carrier was confirmed by liquid chromatography-mass spectrometry. Arthrobacter M3XOD was purified by ammonium sulfate salting out, agarose-guanine affinity chromatography and DEAE-Sepharose CL-4B ion exchange chromatography in three steps. The specific enzyme activity of Arthrobacter M3XOD was 1033.2 U mg-1, the purification multiple was 120.1 and the recovery was 36.1%. The enzymatic properties of Arthrobacter M3XOD showed that XOD contained two subunits (100kDa and 35kDa). The heterodimer protein, with relative molecular weight of 135 kDa, was well matched with Arthrobacter sp. FB24XDH (peptide mass fingerprint analysis); the optimum reaction temperature was 37, and the optimum reaction pH was 7.5. The tolerance to different metal ions (2.0 mmol L-1) was different, and the kinetic constant of xanthine was 0.67 mmol L-1. The XOD enzymes were analyzed at different time. It was found that the thermal instability of XOD was mainly caused by protein aggregation induced by hydrophobic group exposure.
Based on the thermal instability mechanism of XOD, trehalose and betaine were added. The results showed that 1.0mol L-1 trehalose could prolong the half-life of XOD to 6.9h (control group was only 0.84h). The gel filtration chromatography analysis showed that trehalose could inhibit protein aggregation effectively. The thermal stability of XOD was improved by immobilization of lipid (201 *4, D201 and D354), physical immobilization of polyacrylamide and sodium alginate, covalent coupling immobilization of hydrophobic carrier (D840) and hydrophilic carrier (containing agarose with different spacer arm lengths). The relative enzyme activity retention rate of the immobilized enzyme covalently coupled with agarose-ethylenediamine carrier was higher (90.9%). Compared with the free enzyme, the tolerance of the immobilized enzyme at different pH and temperature was improved, and the half-life of the immobilized enzyme was prolonged to 5.5 h (50 C) after repeated use for 8 times. Relative enzyme activity still retained 41.2%.
The relationship between the key metabolites (free radicals, uric acid, urea and ammonium ions) in the hypoxanthine/xanthine degradation pathway and the synthesis of XOD was studied. It was found that ammonium ion, the nitrogen-containing end product of purine metabolism, was the key factor to inhibit the synthesis of XOD during XOD fermentation. In order to reduce the degradation of urea to ammonium ion in the hypoxanthine/xanthine degradation metabolic pathway, a mutant Arthrobacter M605 with low urea degrading enzyme production was semi-directionally bred by plasma mutagenesis and preliminary screening on a three-stage screening plate. The average yield of urea degrading enzyme of Arthrobacter M3 was 49.3% lower than that of Arthrobacter M3. The average yield of XOD and enzyme activity of mutant Arthrobacter M60 5 were 116.5 U g-1 and 12970.6 U L-1, respectively, 38.6% and 42.5% higher than that of the original strain Arthrobacter M3.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：TQ925

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