节杆菌黄嘌呤氧化酶生物合成调控与酶稳定性研究
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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%。
As one of the flavoprotein oxidases, xanthine oxidase (EC1.17.3.2, XOD) has acomplicate quaternary structure and contains multi-cofactors including one molybdenumcofactor,[2Fe–2S] clusters and one FAD cofactor. The above cofactors sequentially act oncatalyzing purine degradation with oxygen. The application of XOD is wider than that ofxanthine dehydrogenase (EC1.17.1.4, XDH) in the field of medical diagnosis, food detection,industrial catalysis, and environmental protection.
In this dissertation, the XOD-producing strain (Arthrobacter M3) with independentintellectual property rights was employed. First, fermentation control protocols to improveXOD average yield were investigated. Then, the affinity medium was synthesized withguanine as ligand, and the simple affinity protocol for purification of XOD was explored. Themechanism of XOD thermal instability from Arthrobacter M3was analyzed. Some targetedapproaches were discussed to improve XOD stability. Finally, plasma mutagenesis wasemployed to semi-directionally screen a mutant strain with the inhibitional attenuation ofmetabolite in purine degradation. The main results are listed as follows,
It was found that the optimum additive concentration of hypoxanthine (inducer) was3.6g L-1. The average yields of XOD by adding riboflavin (0.30mg L-1) and thiamine (6.0mg L-1)were17.0%and16.3%higher than those without adding these two substances. Based on theanalysis of kinetic parameters under various pH conditions, a pH-shift strategy in batchfermentation was implemented to enhance XOD biosynthesis. In this strategy, the initialculture pH was set at8.6without control, then pH was maintained at7.6after the biomassreached2.0g L-1. Compared with results from constant pH fermentation (pH7.6) anduncontrolled pH fermentation (initially at pH8.6), XOD average yield (1229.7U g-1) usingthis pH-shift strategy increased by86.3%and89.4%, and its production (7415.3U L-1)increased by75.0%and91.0%, respectively. Then, Logistic equation and Luedeking-Piretequation were introduced to describe cell growth, XOD accumulation, and substrateconsumption. The correlation coefficients (R2) of these models were all more than0.97.
The result of adsorption analysis showed that Sepharose-guanine affinity medium (2.0mg g-1medium) had the optimum adsorption to XOD among the affinity mediumssynthesized, moreover, the successful coupling of guanine to Sepharose medium was furtherconfirmed by high performance liquid chromatography-mass spectrometry (HPLC-MS). Thesimple protocol for XOD purification was developed, which consisted of only three steps(ammonium sulfate precipitation, affinity extraction, and DEAE ion-exchangechromatography). The specific activity, purification fold, and activity recovery of purifiedXOD were1033.2U mg-1,120.1, and36.1%, respectively. XOD from Arthrobacter M3wasdetermined as a heterodimer containing two subunits (100kDa and35kDa) with a molecularweight of135kDa. The optimal reaction temperature and pH for XOD are37℃and7.5,respectively. The tolerances of XOD to various metals (2.0mmol L-1) are different. The Kmvalue of XOD for xanthine is0.67mmol L-1. The mechanism of XOD thermal instability fromArthrobacter M3mainly involves the exposure of hydrophobic residues and formation of aggregates (50℃).
Based on the above mechanism of XOD thermal instability from Arthrobacter M3, itsstability was significantly enhanced by the addition of corresponding additives such astrehalose (cosolute) and betaine (osmolyte). Preferably, the half-life of XOD activity in thepresence of1.0mol L-1trehalose was increased to6.9h, and the control was only0.84h(50℃). The profiles of SEC about samples with trehalose further confirmed the inhibitioneffect of trehalose on XOD aggregation. On the other hand, ion exchange resins (201×4, D201,D354), polyacrylamide, sodium alginate, the hydrophobic carrier (D840), and hydrophiliccarriers with different spacer length were employed to immobilize XOD. Compared with theabove methods, the optimum activity recovery of immobilization was achieved by usingsodium alginate (17.3%); after incubation at50℃for2h, the relative activity of XODimmobilized onto the Sepharose-ethanediamine carrier (Sepharose-ethanediamine-XOD,90.9%) was shown to be optimum. Compared with the free XOD, Sepharose-ethanediamine-XOD had more favorable tolerance for acid-alkali and temperature, and half-life of thisimmobilized XOD increased to5.5h at50℃. Sepharose-ethanediamine-XOD still kept41.2%of initial activity after it was repeatedly used8times.
The effects of metabolites (free radicals, uric acid, urea, and ammonium) inhypoxanthine/xanthine degradation pathway on XOD biosynthesis were first investigated, andit was found that ammonium (the end product) was the key factor inhibiting XODbiosynthesis. However, urea accumulated in batch fermentation (1.10g L-1) almost had noeffect on XOD biosynthesis, moreover, this concentration (1.10g L-1) was far below the ureaconcentration inhibiting XOD biosynthesis (≥15.0g L-1). In order to attenuate the degradationof urea to ammonium, the mutant strain (Arthrobacter M605) producing low urea-degradingenzyme was screened semi-directionally by plasma mutagenesis. Compared with the result bythe starting strain Arthrobacter M3, the average yield of urea-degrading enzyme by themutant strain Arthrobacter M605decreased by49.3%. XOD average yield (1168.5U g-1) andproduction (12970.6U L-1) by Arthrobacter M605were38.6%and42.5%higher than thoseby Arthrobacter M3in fed-batch fermentation.
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%。
As one of the flavoprotein oxidases, xanthine oxidase (EC1.17.3.2, XOD) has acomplicate quaternary structure and contains multi-cofactors including one molybdenumcofactor,[2Fe–2S] clusters and one FAD cofactor. The above cofactors sequentially act oncatalyzing purine degradation with oxygen. The application of XOD is wider than that ofxanthine dehydrogenase (EC1.17.1.4, XDH) in the field of medical diagnosis, food detection,industrial catalysis, and environmental protection.
In this dissertation, the XOD-producing strain (Arthrobacter M3) with independentintellectual property rights was employed. First, fermentation control protocols to improveXOD average yield were investigated. Then, the affinity medium was synthesized withguanine as ligand, and the simple affinity protocol for purification of XOD was explored. Themechanism of XOD thermal instability from Arthrobacter M3was analyzed. Some targetedapproaches were discussed to improve XOD stability. Finally, plasma mutagenesis wasemployed to semi-directionally screen a mutant strain with the inhibitional attenuation ofmetabolite in purine degradation. The main results are listed as follows,
It was found that the optimum additive concentration of hypoxanthine (inducer) was3.6g L-1. The average yields of XOD by adding riboflavin (0.30mg L-1) and thiamine (6.0mg L-1)were17.0%and16.3%higher than those without adding these two substances. Based on theanalysis of kinetic parameters under various pH conditions, a pH-shift strategy in batchfermentation was implemented to enhance XOD biosynthesis. In this strategy, the initialculture pH was set at8.6without control, then pH was maintained at7.6after the biomassreached2.0g L-1. Compared with results from constant pH fermentation (pH7.6) anduncontrolled pH fermentation (initially at pH8.6), XOD average yield (1229.7U g-1) usingthis pH-shift strategy increased by86.3%and89.4%, and its production (7415.3U L-1)increased by75.0%and91.0%, respectively. Then, Logistic equation and Luedeking-Piretequation were introduced to describe cell growth, XOD accumulation, and substrateconsumption. The correlation coefficients (R2) of these models were all more than0.97.
The result of adsorption analysis showed that Sepharose-guanine affinity medium (2.0mg g-1medium) had the optimum adsorption to XOD among the affinity mediumssynthesized, moreover, the successful coupling of guanine to Sepharose medium was furtherconfirmed by high performance liquid chromatography-mass spectrometry (HPLC-MS). Thesimple protocol for XOD purification was developed, which consisted of only three steps(ammonium sulfate precipitation, affinity extraction, and DEAE ion-exchangechromatography). The specific activity, purification fold, and activity recovery of purifiedXOD were1033.2U mg-1,120.1, and36.1%, respectively. XOD from Arthrobacter M3wasdetermined as a heterodimer containing two subunits (100kDa and35kDa) with a molecularweight of135kDa. The optimal reaction temperature and pH for XOD are37℃and7.5,respectively. The tolerances of XOD to various metals (2.0mmol L-1) are different. The Kmvalue of XOD for xanthine is0.67mmol L-1. The mechanism of XOD thermal instability fromArthrobacter M3mainly involves the exposure of hydrophobic residues and formation of aggregates (50℃).
Based on the above mechanism of XOD thermal instability from Arthrobacter M3, itsstability was significantly enhanced by the addition of corresponding additives such astrehalose (cosolute) and betaine (osmolyte). Preferably, the half-life of XOD activity in thepresence of1.0mol L-1trehalose was increased to6.9h, and the control was only0.84h(50℃). The profiles of SEC about samples with trehalose further confirmed the inhibitioneffect of trehalose on XOD aggregation. On the other hand, ion exchange resins (201×4, D201,D354), polyacrylamide, sodium alginate, the hydrophobic carrier (D840), and hydrophiliccarriers with different spacer length were employed to immobilize XOD. Compared with theabove methods, the optimum activity recovery of immobilization was achieved by usingsodium alginate (17.3%); after incubation at50℃for2h, the relative activity of XODimmobilized onto the Sepharose-ethanediamine carrier (Sepharose-ethanediamine-XOD,90.9%) was shown to be optimum. Compared with the free XOD, Sepharose-ethanediamine-XOD had more favorable tolerance for acid-alkali and temperature, and half-life of thisimmobilized XOD increased to5.5h at50℃. Sepharose-ethanediamine-XOD still kept41.2%of initial activity after it was repeatedly used8times.
The effects of metabolites (free radicals, uric acid, urea, and ammonium) inhypoxanthine/xanthine degradation pathway on XOD biosynthesis were first investigated, andit was found that ammonium (the end product) was the key factor inhibiting XODbiosynthesis. However, urea accumulated in batch fermentation (1.10g L-1) almost had noeffect on XOD biosynthesis, moreover, this concentration (1.10g L-1) was far below the ureaconcentration inhibiting XOD biosynthesis (≥15.0g L-1). In order to attenuate the degradationof urea to ammonium, the mutant strain (Arthrobacter M605) producing low urea-degradingenzyme was screened semi-directionally by plasma mutagenesis. Compared with the result bythe starting strain Arthrobacter M3, the average yield of urea-degrading enzyme by themutant strain Arthrobacter M605decreased by49.3%. XOD average yield (1168.5U g-1) andproduction (12970.6U L-1) by Arthrobacter M605were38.6%and42.5%higher than thoseby Arthrobacter M3in fed-batch fermentation.
引文
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