大豆过氧化物酶的酶学特性与应用研究

【作者】 徐芝勇；

【导师】 张建国；

【作者基本信息】 浙江大学 ， 生物物理学， 2006， 硕士

【摘要】 大豆过氧化物酶是从大豆加工副产物大豆皮中提取的,以血红素为辅基的Ⅲ类过氧化物酶(EC1.11.1.7)。与辣根过氧化物酶在结构和功能上都有很多相似之处,但因其原料价廉易得,底物作用范围广、耐热性能高、酸碱稳定性好、pH适用范围宽等优点,大豆过氧化物酶的研究越来越成为过氧化物酶研究的热点。大豆过氧化物酶在食品加工、生物医学检测、农产品贮藏、污水处理和木质素降解及生物合成方面的应用研究已经广泛展开。尤其在免疫诊断试剂术、生物传感器、木素降解和工业“三废”处理等方面的应用研究进展飞速。所以大豆过氧化物酶很可能会成为辣根过氧化物酶、木质素过氧化物酶等过氧化物酶一个最有竞争力的替代品,成为过氧化物酶的一个很好的来源。大豆过氧化物酶这些特殊的性质使其具有广阔约应用前景。 大豆过氧化物酶是一种由单一肽链与卟啉构成的血红素蛋白,脱辅基蛋白分子与血红素结合构成全酶。大豆过氧化物酶的分子量约为39～41kD,由306个氨基酸残基组成,并含有大约分子量的18%的糖基。其等电点为3.9,属于酸性蛋白质。 本文在实验室对大豆过氧化物酶分离纯化研究的基础上,对自己纯化所得的酶进行酶学特性的研究。结果表明,大豆过氧化物酶的温度和pH适用范围很宽,热稳定性、酸碱稳定性很高,并且在含有较高浓度有机溶剂的反应体系中的保持很好的酶活力,同时研究了一般常见化合物对酶活性的影响,以及测定了部分酶促反应动力学参数。 同时本文开展了大豆过氧化物酶的应用研究。选取大豆过氧化物酶在免疫诊断试剂中的应用为研究的突破口。先制备大豆过氧化物酶与抗体的结合物,再把结合物用于酶联免疫吸附测定,采用双抗体夹心法,检测目标抗原的浓度。用此方法来证明自己纯化的大豆过氧化物酶用于酶联免疫吸附测定的可行性,并比较大豆过氧化物酶与抗体的结合物和辣根过氧化物酶与抗体的结合物在检测抗原中的异同。初步结果表明,大豆过氧化物酶本身有很多特性非常适用于免疫诊断试剂,并已经发现在部分指标上优于辣根过氧化物酶,如果能结合大豆过氧化物酶的特点进一步优化方案,将来大规模应用于诊断试剂的生产可能性很大。更多还原

【Abstract】 Soybean peroxidase (SBP) obtained from the soybean coats belongs to class III of the plant peroxidase superfamily(EC1.11.1.7), similar to horseradish peroxidase (HRP). SBP is very stable at high temperature, extremes of pH, and in organic solvent. At the same time, it is highly reactive towards both organic and inorganic substrates, similar to horseradish peroxidase. SBP has a wide range of potential applications,and its structure is of particular interest for engineering purposes and as a model for stable heme peroxidases. SBP has been found to be a very effective of biocatalyst and biosensor which has been used in wastewater treatment and phenolic resin synthesis.SBP is a highly heterogeneous glycoprotein with MS determined masses from 39 to 41 kDa. The mature protein consists of 306 residues starting with pyrrolidone carboxylic acid. SBP is glycosylated corresponding to approximately 1.8% of the molecular mass, and the glycans are known to be heterogeneous.A brief protocol for purification of SBP was worked out in our previous work. Now, a systematic evaluation of the biocatalytic properties of SBP was carried out in this study. The results shown that SBP is able to retain its catalytic activity under wide of pH and at elevated temperatures. Furthermore, SBP is very stable at high temperature, extremes of pH and in organic solvents. We also measured the apparent Michaelis constant (Km) of SBP for the oxidation of guaiacol by hydrogen peroxide.Soybean peroxidase is similar to horseradish peroxidase, a popular enzyme for generating enzyme-labeled conjugates. SBP can be used to generate enzyme conjugates with superior thermostability, pH stability and stability in organic solvents over HRP. In this study, we labeled antibody molecules with SBP other than HRP using the most common method. Then we evaluated the antibody-enzyme complex by enzyme linked immunosorbent assay (ELISA).In summary, we observed that SBP possesses substantially high catalytic efficiency and conformational stability, and remains stable over a wide pH range and at high temperature. These superior qualities of SBP may, in the future, help it to replace HRP in diagnostic, biosensor, and biotechnological uses, especially in wastewater treatment.更多还原