節(jié)桿菌黃嘌呤氧化酶生物合成調(diào)控與酶穩(wěn)定性研究
發(fā)布時間：2018-09-07 18:43
【摘要】：黃嘌呤氧化酶（Xanthine oxidase，EC1.17.3.2，XOD）屬于黃素蛋白氧化酶類中較為復雜的多亞基蛋白，其底物催化機理復雜，除需輔因子FAD，還需鉬蝶呤及鐵硫簇輔因子。這三種輔因子以嚴格的比例及特定的順序排布于XOD結構內(nèi)部，聯(lián)合最終電子受體（分子氧），，共同催化嘌呤類物質(zhì)的降解。相比于黃嘌呤脫氫酶（Xanthinedehydrogenase，EC1.17.1.4，XDH），XOD在醫(yī)學診斷、食品檢測、工業(yè)催化及環(huán)境保護中的應用價值更為廣泛。 本論文以一株擁有自主知識產(chǎn)權的XOD產(chǎn)生株—節(jié)桿菌（Arthrobacter）M3為研究對象，探討發(fā)酵合成XOD的調(diào)控技術；合成篩選含特異性配體的親和介質(zhì)，創(chuàng)建簡易親和純化XOD的方法，并分析酶熱不穩(wěn)定機理；探尋可有效提高XOD穩(wěn)定性的技術；研究次黃嘌呤/黃嘌呤降解代謝產(chǎn)物與XOD合成的關系，利用等離子體誘變，半定向篩選代謝產(chǎn)物抑制得到衰減的突變株。主要研究結果如下： 考察次黃嘌呤（誘導物）及輔因子添加對XOD合成的影響，發(fā)現(xiàn)，次黃嘌呤最佳誘導濃度為3.6g L-1，而添加輔因子前體核黃素（0.30mg L-1）及硫胺素（6.0mg L-1）可分別使XOD平均產(chǎn)率提高17.0%和16.3%。根據(jù)不同pH值控制下Arthrobacter M3的發(fā)酵過程曲線和動力學參數(shù)變化，提出了分段式pH調(diào)控技術，即在發(fā)酵前期以初始pH8.6進行自然發(fā)酵，待菌體密度至2.0g L-1時，控制發(fā)酵液pH值為7.6。該分段式pH調(diào)控技術的應用，使XOD平均產(chǎn)率（1229.7U g-1）比單獨使用恒pH7.6發(fā)酵和自然pH發(fā)酵（初始pH8.6）時分別提高了86.3%和89.4%；酶活水平（7415.3U L-1）分別提高了75.0%和91.0%。以Logistic方程和Luedeking-Piret方程描述了分段式pH發(fā)酵過程中菌體生長、XOD積累以及基質(zhì)（殘?zhí)牵┫牡哪Ｐ停Ｐ拖嚓P系數(shù)（R2）均大于0.97。 分別以鳥嘌呤（黃嘌呤結構類似物）及核黃素（FAD前體）為配體，合成了親和介質(zhì)（瓊脂糖為載體）。吸附分析表明，瓊脂糖-鳥嘌呤親和介質(zhì)對XOD的吸附能力較佳（2.0mg g-1介質(zhì)），并利用液質(zhì)聯(lián)用技術證實了鳥嘌呤配體與瓊脂糖載體的成功偶聯(lián)。采用硫酸銨鹽析、瓊脂糖-鳥嘌呤親和層析及DEAE-Sepharose CL-4B離子交換層析共3步法，簡易地純化了Arthrobacter M3XOD，比酶活為1033.2U mg-1，純化倍數(shù)為120.1，回收率為36.1%。研究Arthrobacter M3XOD的酶學性質(zhì)則表明：XOD為含有兩個亞基（100kDa和35kDa）的異質(zhì)二聚體蛋白，相對分子質(zhì)量135kDa，與Arthrobacter sp. FB24XDH的匹配度較高（肽質(zhì)量指紋圖譜分析）；最適反應溫度為37℃，最適反應pH為7.5，且對不同金屬離子（2.0mmol L-1）的耐受性具有差異，對黃嘌呤的動力學常數(shù)Km為0.67mmol L-1。對50℃保溫不同時間的XOD酶液進行分析，發(fā)現(xiàn)XOD熱不穩(wěn)定機理主要為疏水基團暴露引起的蛋白集聚。 以XOD的熱不穩(wěn)定機理為指導，針對性的添加了海藻糖和甜菜堿等保護劑。結果表明，添加1.0mol L-1海藻糖可使XOD半衰期（50℃）延長至6.9h（對照組僅為0.84h），凝膠過濾色譜分析表明海藻糖的加入有效抑制了蛋白集聚。分別選用陰離子交換樹脂（201×4、D201及D354）離子吸附固定化、聚丙烯酰胺及海藻酸鈉物理包埋固定化、疏水性載體（D840）及親水性載體（含不同間隔臂長度的瓊脂糖）共價偶聯(lián)固定化處理XOD，不同程度提高了酶熱穩(wěn)定性。其中，以海藻酸鈉物理包埋的固定化酶酶活回收率較高（17.3%）；50℃保溫2h，以瓊脂糖-乙二胺載體共價偶聯(lián)的固定化酶相對酶活保留率較高（90.9%）。將瓊脂糖-乙二胺載體固定化的酶與游離酶比較，固定化酶于不同pH和溫度下的耐受性均提高，半衰期延長至5.5h（50℃），重復使用8次后，相對酶活仍保留41.2%。 研究次黃嘌呤/黃嘌呤降解代謝途徑中關鍵代謝產(chǎn)物（自由基、尿酸、尿素及銨根離子）與XOD合成的關系，發(fā)現(xiàn)，在XOD發(fā)酵合成過程中，嘌呤代謝含氮終產(chǎn)物—銨根離子是抑制XOD合成的關鍵因子；積累的代謝中間產(chǎn)物尿素對XOD合成基本無影響，且積累濃度（1.10g L-1）遠低于其抑制XOD合成的濃度（≥15.0g L-1）。為減弱次黃嘌呤/黃嘌呤降解代謝途徑中尿素向銨根離子的降解，采用等離子體誘變，經(jīng)三級篩選平板初篩，半定向選育了一株低產(chǎn)尿素降解酶的突變株Arthrobacter M605，與出發(fā)菌株Arthrobacter M3相比，其尿素降解酶平均產(chǎn)率降低了49.3%。補料分批發(fā)酵條件下，突變株Arthrobacter M605的XOD平均產(chǎn)率及酶活水平提高至1168.5U g-1和12970.6U L-1，分別比出發(fā)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.
【學位授予單位】：江南大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：TQ925
本文編號：2229104
[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.
【學位授予單位】：江南大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：TQ925
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