【作者】 李忠琴；

【导师】 王武；

【作者基本信息】 江南大学 ， 发酵工程， 2007， 博士

【摘要】 黄嘌呤氧化酶（xanthine oxidase，EC1．2．3．22）是嘌呤代谢途径的关键酶，广泛分布于各种生物体的组织和细胞中，主要催化次黄嘌呤氧化生成黄嘌呤，进一步氧化黄嘌呤生成尿酸。临床上可用于制备免疫抗体、肿瘤抑制剂和细胞缺血性再灌注损伤等疾病的检测。根据辣根过氧化物酶和黄嘌呤氧化酶的催化反应特性，本文提出了以偶联辣根过氧化物酶-苯酚-4-氨基安替比林反应显色，检测黄嘌呤氧化酶活力的新方法。最佳测定条件为：辣根过氧化物酶7000U·L^-1，4-氨基安替比林1 mmol·L^-1，苯酚6 mmol·L^-1，黄嘌呤1 mmol·L^-1溶于50 mmol·L^-1Tris-HCL缓冲液（pH8．4）；反应温度为37℃，保温时间为20min；检测波长为508nm。本方法测定XOD酶活的线性范围为5．0～100．0U·L^-1，线性关系良好（r=0．9992），检出限为1．3U·L^-1。以黄嘌呤为唯一碳氮源，采用富集分离培养方法，在国内首次筛选获得一株产黄嘌呤氧化酶细菌。经生理生化鉴定和16S rDNA序列同源分析，确定其属于微球菌科节杆菌属，命名为Arthrobactersp．XL26，GenBank接受号为EF532600。此菌在种子培养基和发酵基本培养基中的生长情况表明，达到最高菌体生长量的培养时间分别为24h和42h，发酵培养45h可达最高产酶活力，表明该菌发酵合成黄嘌呤氧化酶基本属于生长偶联型。通过单因素扫描、Plackett-Burman、Box-Benken试验设计和响应面分析法优化了产酶发酵条件和培养基组成。最适发酵条件为：初始培养基pH8．5，培养温度32℃，接种量10％，摇瓶培养基装液比例30％，转速210 r·min^-1；最适培养基组成为(g·L^-1)：乳糖：葡萄糖（3：1）12．0，（NH₄）₂SO₄ 4．0，酵母粉2．13，黄嘌呤2．57，FeSO₄·7H₂O 0．5×10^-3，MgSO₄·7H₂O 0．2×10^-3，CaCl₂ 5．33×10^-3，KH₂PO₄ 0．94，Na₂HPO₄ 5．92。优化后每升发酵液产XOD可达409．6U，而且比酶活比初始基本发酵提高了15倍。进一步采用间歇补料，产酶量又增加了35％。经过超声破碎、硫酸铵分级盐析、PEG6000／（NH₄）₂SO₄双水相萃取、Butyl-Sepharose 4B疏水层析、DEAE Sepharose CL-6B FF离子交换、Phenyl Sepharose CL-4B疏水层析和Sephacryl S-200 HR凝胶过滤，从Arthrobacter sp．XL26中分离得到黄嘌呤氧化酶，其比酶活提高了270倍，达24 U·mg^-1，回收率为11％。SDS-PAGE确定此蛋白含有两个大小不等的亚基，相对分子量分别约为55．48kDa和85．67 kDa。推断该酶为两个不同亚基构成的二聚体分子（α₂β₂）。酶学性质研究表明，来源于Arthrobacter sp．XL26的黄嘌呤氧化酶最适反应pH为8．0，最适温度为55℃；在pH7．0-9．0 Tris-HCl缓冲液中于25℃保温16h，剩余相对酶活高于80％；在35℃以下保温30min，酶活保持100％。以黄嘌呤和次黄嘌呤为底物的表观米氏常数分别为0．032mmol·L^-1和0．133mmol·L^-1。金属离子Cu²⁺和pb²⁺对XOD有强烈的抑制作用，Ag⁺和Hg²⁺完全抑制了酶活性；而Mn²⁺、Co²⁺和Ba²⁺对XOD的酶活性略有激活作用。化学物质中水杨酸钠、组胺、酒石酸钾钠、甘露醇、丙三醇、二硫苏糖醇、聚乙烯吡咯烷酮（PVP）、聚乙二醇（PEG）6000及表面活性剂Triton X-114对XOD酶均为XOD的激活剂，可显著提高酶反应的速率；叠氮钠和尿素可强烈抑制XOD酶活性；山梨醇和甘氨酸可以极大地提高酶热稳定作用。根据GenBank、EMBL和SWISS-PROT报导的黄嘌呤氧化酶蛋白保守序列，设计简并引物，以Arthrobacter sp．XL26基因组为模板，采用简并PCR、反向PCR和巢式PCR技术扩增获得编码黄嘌呤氧化酶A、B亚基基因的全长序列分别为1542bp和2355bp，Genbank上的登录号分别为EF648005和EF648204，推测其各编码513个氨基酸残基的A亚基肽链和784个氨基酸残基的B亚基肽链。同源性比对显示，xodA和xodB推导的氨基酸序列分别与来源于Acinetobacter baumannii ATCC 17978的黄嘌呤脱氢酶小亚基和大亚基同源性达到了92％和84％。蛋白质结构在线分析表明，Arthrobactersp．XL26黄嘌呤氧化酶的A、B亚基均属于（α+β）型蛋白，其中[2Fe-2S]结合位点和FAD结合域位于XODA肽链，而XODB肽链则含有钼蝶呤结合域及醛氧化酶/黄嘌呤脱氢酶特有的a／b锤头结合域。更多还原

【Abstract】 Xanthine oxidase （XOD, EC 1.2.3.22） is a complex molybdoflavoenzyme that exists in as an available oxidoreductase in all kinds of organisms. It is also a key enzyme in the catabolism of purines path way, which mainly catalyzes the conversion of hypoxanthine and xanthine to xanthine and uric acid, respectively. The enzyme has broad substrate specificity, except catalyzing the oxidation of purine, pyrimidine, pterine, and aldehyde, it catalyses 7,8-dihydropterin to 7,8-dihydroxanthopterin, xanthopterin to leucopterin, and pterin to isoxanthopterin or drosopterin as well. It has been clinically used as anti-tumor and clinical diagnosis of reperfusion injury.A novel method to determine the activity of xanthine oxidase through the chromogenic reaction of 4 -aminoantipyrine （AAP）, phenicacid （PA） and hydrogen peroxide, which was produced via the oxidation of xanthine catalyzed by XOD, under the help of horseradish peroxidase （HRP）, was proposed. The influences of temperature、pH and amount of substrate on the activity of XOD were investigated. The optimal conditions for XOD activity assay were obtained as follows: HRP (7000U·L^-1), AAP (1mmol·L^-1), PA (6mmol·L^-1) and xanthine (1mmol·L^-1) were dissolved in 50 mmol·L^-1 Tris-HCl buffer solution （pH8.4）, the reaction temperature was 37℃, the reaction time was 20 min, the detective wavelength was 508nm. Under the conditions mentioned above, the linear range of calibration was between 5.0 U·L^-1 and 100.0 U·L^-1, and the detection limit was 1.3 U·L^-1. The above system is a potential alternative method to determine the activity of XOD in the areas either for laboratory assay or clinical diagnosis.Using xanthine as sole carbon, nitrogen, energy sources and inductor, a xanthine oxidase producer was isolated from the soil. The strain was characterized not only by morphologic and biochemiccal properties, but also by clone and sequence of its 16S rDNA. As a result, the organism has been identified as Arthrobacter and named Arthrobacter sp.XL26 whose GenBank acceptor number is EF532600.The effects of inductor, carbon and nitrogen sources on XOD production by Arthrobacter sp. XL26 were investigated. The medium required for XOD production was optimized with statistics based experimental designs. The important medium components, identified by initial screening method of Plackett-Burman, were xanthine, yeast extract and CaCl₂. Box-Behnken and response surface methodology were then employed to further optimize XOD production. The optimal compositions for higher production of XOD were (g·L^-1): glucose 12.0, yeast extract 2.13, xanthine 2.57, （NH₄）₂SO₄ 4.0, KH₂PO₄.3H₂O 0.94, Na₂HPO₄ 5.92; CaCl₂ 5.33×10^-3, MgSO₄.7H₂O 0.2×10^-3, FeSO₄.7H₂O 0.5×10^-3. Using this statistical optimization method, the XOD was found to increase from 27.4 U·L^-1 to 409.6 U·L^-1. XOD has been purified to a single protein band from cell-free extracts of Arthrobacter sp. XL26 via procedures of ultrasonication, ammonium sulfate fractionation, Butyl-Sepharose 4B chromatography, DEAE Sepharose CL-6B FF chromatography, Phenyl Sepharose CL-4B chromatography and Sephacryl S-200 HR gel filtration. The enzyme specific activity was found to increase to 270-fold and reache 24 U·mg^-1 with a final recovery rate of 11%. The native PAGE and SDS-PAGE show that the enzyme has a molecular weight of 260,000 and consists of two heterogeneous subunits, xodA and xodB, with a molecular weight of 55,480 and 85,670, repectively.The enzyme shows the highest activity at 55℃and pH 8.0. And it is still stable between pH 7 and 9 at 25℃for 16h or below 35℃at pH 8 for 30 min. K_m values of the enzyme for hypoxanthine and xanthine are0.133 and 0.032 mM, respectively. The activity can be stimulated by Mn²⁺, Co²⁺and Ba²⁺, and inhibited by Fe²⁺, Cu²⁺, Pb²⁺, Ag⁺and Hg²⁺ at a concentration of 2mM or more. Among the compounds tested, sodium salicylate, histamine, triton, tween 80, rochelle salt, mannitol, sorbitol, glycerine, PVP, DTT and PEG6000 are activity stimulator. The activity can be strongly inhibited by NaN₃ and urea as well.The xodA and xodB from Arthrobacter sp. XL26, encoding the heterodimeric molybdo-iron-sulfur-protein xanthine oxidase, were cloned and sequenced by the technology of inverse PCR and nest PCR. The results show these two genes consist of 1542 bp and 2355 bp and encode two peptides with 513 and 784 amino acids, whose sequences acceptor number in GenBank are EF648005 and EF648204 respectively. The deduced amino acid sequences of xodA and xodB show homologies of 92% and 84% comparing to the small and large subunits of Acinetobacter baumannii ATCC 17978 xanthine dehydrogenases, respectively. Two-dimensional structure models indicate its [2Fe-2S] and FAD-binding domains locate in xodA, whereas Mo-binding and a/b hammer-head domain of aldehyde oxidase or xanthine dehydrogenase seat in xodB.更多还原