【作者】 金朝霞；

【导师】 王长海； 李宪臻；

【作者基本信息】 大连理工大学 ， 生物化工， 2008， 博士

【摘要】 草酸盐降解菌及其降解酶的研究具有很多潜在的应用价值。一方面可用于致病真菌Sclerotinia sclerotiorum所引起的重要经济作物菌核病的防治，及抗病性转基因植物的生产：另一方面，草酸盐降解酶被用于诊断和治疗与草酸盐相关的疾病，如肾结石，以及环境的生物治理。本论文分离筛选出具有较强草酸盐降解能力的新菌，并对该菌降解酶的酶学性质及降解机理进行了研究。在酸性介质中草酸能够催化重铬酸钾对次甲基蓝的氧化脱色反应，这种催化效率与草酸浓度呈正相关，据此建立了次甲基蓝-重铬酸钾体系测定草酸的方法。实验证明，在660 nm处不加草酸与加草酸的次甲基蓝-重铬酸钾体系反应后的吸光度差值△A在0～80μg／mL范围内与草酸浓度呈线性关系，线性方程为：△A=0．0984ρ_草酸-0．0044，相关系数为0．9986。反应的最适条件为3．752×10^-5mol／L次甲基蓝、0．12mol／L H₂SO₄和2×10^-3mol／L K₂Cr₂O₇。以草酸盐为唯一碳源和能源，从土壤中分离筛选获得一株能够降解草酸及其盐类的细菌，命名为OXJ-11。该菌在形态培养基形成的菌落为乳白色，圆形；革兰氏阴性染色为阴性杆菌；经电镜观察，为端生双鞭毛。接触酶阳性：不还原硝酸盐。能够在温度25℃-35℃范围内和pH6．0-10．0范围内好氧生长。根据其形态特征、生理生化特征及16SrRNA序列同源性分型，OXJ-11为Pandoraea属的一株新菌。这种菌能够合成草酸盐降解酶。OXJ-11产酶的最适发酵培养基配方为：0．5％草酸钠，0．3％酵母浸粉，0．05％MgSO₄·7H₂O，最适温度为30℃，pH为6．0-7．0。Pandoraea sp.OXJ-11的草酸盐降解酶为底物草酸盐诱导酶。以次甲基蓝-重铬酸钾氧化还原法测定反应体系草酸盐的减少量，建立了草酸盐降解酶的酶促反应体系。该酶为胞壁酶，超声破碎的条件为：输出功率200w-300w，超声破碎0．5s、间歇5s，破碎总时间7min。草酸盐降解酶的最适初始底物浓度为0．7mg／mL；最适反应温度为30℃；30℃下保温酶活力较稳定；酶反应的最适pH范围较宽为5．5-8．0；粗酶液在pH7．0-8．0范围内保温比较稳定；不同浓度的Fe²⁺和Cr³⁺对酶均有抑制作用；SDS对酶有很强的抑制作用；10mmol／L的Mg²⁺对酶有一定的激活作用；NaCl对酶活力有较大的影响，0．15mol／L NaCl可使酶活力下降约50％，浓度达到0．3mol／L时酶活力完全丧失。草酸盐降解酶的酶解产物之一为二氧化碳，当反应体系中含有阳离子时，二氧化碳会与阳离子反应生成碳酸盐。而过氧化氢不是酶反应产物。甲酸盐为酶解反应的另一产物，但由于OXJ-11菌中含有甲酸盐脱氢酶，因此，甲酸盐被进一步降解生成二氧化碳。OXJ-11菌的草酸盐降解酶所催化的反应，必须在有氧条件下才能进行。草酸和琥珀酸为草酸盐降解酶的可替换底物。ATP和ADP对草酸钠降解酶活性均有不同程度的抑制作用。更多还原

【Abstract】 Oxalate-degrading strains and oxalate-degrading enzymes have a number of potential applications, including the control of sclerotinia stem rot in economically important plants caused by fungus Sclerotinia sclerotiorum, and in making plants resistant to the white mold disease by oxalate-degrading enzyme transgene, including medical diagnosis and treatments for hyperoxaluria and other oxalate-related diseases, bioremediation of the environment. The paper is to isolate the bacteria that can degrade oxalate, study degrading enzyme characteristics and the degrading mechanism.A method for the determination of oxalic acid with methylene blue - dichromate system had been developed. It was based on the catalytic effect of oxalic acid on the oxidation of methylene blue by dichromate in acidic media. The absorbance of methylene blue at the maximum wavelength of 660 nm was recorded at fixed reaction times. The absorbance differences （△A ） of the methylene blue - dichromate system with and without oxalic acid were linearly correlated with the oxalic acid concentration. The linear range of the calibration graph for oxalic acid was 0 - 80μg/mL with a correlation coefficient of 0.9986. The optimum concentrations for methylene blue, H₂SO₄ and K₂Cr₂O₇ were 3.752×10^-5 mol/L, 0.12 mol/L and 2 mmol/L, respectively. The developed method was simple, sensitive and inexpensive.One new strain designated as OXJ-11 was isolated from soil samples, which could grow in the medium with oxalate as the sole carbon and energy source. The bacteria colonies on plate appeared milky, circular and the isolate OXJ-11 was Gram-negative straight rod. It occured singly and was motile by means of a double polar flagellum. Catalase was positive and nitrate was not reduced. It growed aerobically at 25℃-30℃and pH 6.0 - 10.0, respectively. The polyphasic taxonomic data along with 16S rRNA sequence comparison demonstrated that the isolate OXJ-11 should belong to the genus Pandoraea and represented a new member in this family. The optimal fermentation medium composition was 0.5% sodium oxalate, 0.3% yeast extract powder, 0.05% MgSO₄·7H₂O. And the optimal temperature and pH for the bacterium’s enzyme production were 30℃and pH 6.0-7.0. Pandoraea sp.OXJ-11 had been shown to produce the degrading enzyme which could be induced by the oxalate in medium. Quantitating the reduction of oxalate by the methylene blue - dichromate system, a method to assay the oxalate-degrading activity was constructed. The key enzyme was cell wall enzyme, the conditions of ultrasonic cell-break were: output power 200w - 300w, ultrasonic time 0.5s、intermission time 5s, total working time 7min. Tests showed that the optimal initial substrate concentration for oxalate-degrading enzyme was 0.7mg/mL, while the optimal temperature and pH was 30℃and 5.5 - 8.0; the enzyme activity was stable below 30℃and 7.0-8.0. The enzyme activity was inhibited by Fe²⁺ , Cr³⁺ and SDS had strong inhibition on it, while 10mmol/L Mg²⁺ could increase the enzyme activity. 0.15mol/L and 0.3mol/L NaCl reduced the enzyme activity by 50% and 100%, respectively.An enzymatic hydrolysate of the oxalate-degrading was carbon dioxide, and carbonate was formed by reaction between CO₂ and the cation in the system. Hydrogen peroxide was not the enzymatic hydrolysate. Formate was another enzymatic hydrolysate and it was degraded into carbon dioxide by formate dehydrogenase. Oxalate-degrading enzyme in OXJ-11 required molecular oxygen for catalytic turnover. Oxalate-degrading enzyme could use oxalic acid and succinic acid as exchangeable substrate. Both ATP and ADP could inhibit the enzyme activity.更多还原