【作者】 宋耿云；

【导师】 王晓云；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 植酸酶(myo-inostol-hexakisphosphate phosphohydrolase,EC 3.1.3.8)是一种能降解植酸(肌醇六磷酸)为肌醇和磷酸的酶。本文以毕赤酵母表达的Aspergillu sp.植酸酶为材料,主要研究了二硫键对植酸酶复性过程中构象和催化活性的影响、去糖基化对植酸酶稳定性的影响、金属离子对植酸酶活性、构象和稳定性的的影响及底物、产物类似物等抑制动力学。主要研究结果如下: 1.二硫键对植酸酶复性过程中构象和催化活性的影响。利用内源荧光光谱和园二色光谱及酶活性分析技术,研究了脲变性植酸酶在有、无还原剂DTT存在下复性过程中其二级结构、三级结构及催化活性的变化,结果表明:脲变性植酸酶于pH 5.6缓冲液中复性,在有无还原剂DTT存在下其活力均能够恢复到原来活力85%左右:当于pH 8.3缓冲液中复性时,有无DTT的存在对活力恢复的影响较大,其活力能分别恢复到原来的54%和70%。对其CD光谱和荧光光谱的变化分析表明,其空间结构的变化与活力的变化密切相关。说明二硫键在对植酸酶的催化活性和维持空间结构中起着重要作用。 2.去糖基化对植酸酶稳定性的影响。利用糖苷内切酶和高碘酸分别对植酸酶去糖基化处理,研究植酸酶的热稳定性、构象稳定性及抗蛋白酶能力的变化。研究结果表明,去糖基化后植酸酶的热稳定性下降。用糖苷内切酶去糖基化的植酸酶在65℃保温10 min后酶剩余活力大约为35%,用高碘酸去糖基化的植酸酶在同样条件下酶活力完全丧失,而未更多还原

【Abstract】 Phytase (myo-inositol hexakisphate phosphohydrolase, EC 3.1.3.8) catalyze the hydrolysis of phytate to myo-inositol and inorganic phosphate. The influence of disulfide bonds on the conformational changes and activities of refolded phytase, the effect of deglycosylation on the stability of the phytase, the effect of metal on the conformation, activities and stability and inhibition kinetics of substrate, product and product analogues on the phytase was studied using Aspergillus sp. phytase expressed by pichia pastoris as material. By the investigation, the following results were achieved:1. The influence of disulfide bonds on the conformational changes and activities of refolded phytase. In order to elucidate the role of disulfide bonds, this paper dealed the data with intrinsic fluorescence spectra, far-ultraviolet circular dichroism (CD) spectra, and enzyme activity of the reactivation and refolding of phytase in the absence and presence of dithiothreitol (DTT). When the denatured phytase was placed into pH 5.6 refolding solution in the absence and presence of 2 mmol/L DTT, about 85% of the native enzyme activity was recovered. When the enzyme was put into pH 8.3 buffer in the absence and presence of DTT refolding solution, less activity was recovered, about 70% and 54%, respectively. Analyzing the changes of CD spectra and intrinsic fluorescence of refolded phytase, the conformational changes were close to the changes of activities. The results indicated that disulfide bonds played an important role in the catalytic activity and conformational stability.2. The influence of deglycosylation on the stability of phytase. The phytase was deglycosylated with Endo Hf and NaIO4, and its thermal stability, conformational changes and its proteolytic stability was measured. The results indicated that the catalytic activity of deglycosylated phytase decreased apparently than the phytase. The remaining acitivity of deglycosylated phytase with Endo Hf incubated for 10 min at 65 ℃ was about 35%, and the deglycosylated phytase with NaIO4 had no residual activity detected under the same condition. However, its remaining activity of the control was about 45%. Analyzing the intrinsic fluorescence spectra of deglycosylated phytase with NaIO4, the fluorescence emission maximn red shift and the fluorescence intensity decrease apparently. All of these indicated that glycosylation play an important role for its thermal stability. But the proteolytic stability of deglycosated phytase decreased somewhat, it become 90-93% of the phytase.3. The effect of different metal ions on the catalytic activity, conformation and stability was studied. The phytase was activated by 1-4 mmol/L Mg2+, and its relative activity was improved to 117% by 4 mmol/L Mg2+. Its thermal stability was improved 30% if incubated for 30 min at 65 ℃ with 4 mmol/L Mg2+, its conformation changed little from the changes of CD spectra and fluorescence spectra. The phytase was inhibited by 0.1-1.4 mmol/L Cu2+ and 1-10 mmol/L Zn2+ and its inhibiton type was noncompetitive according to Lineweaver-Burk plot, the inhibition constants were 0.51 mmol/L and 1.99 mmol/L respectively. The change of the phytase conformation was not detected by these metal ions from the CD and fluorescence spectra.4. The effects of a substrate, a product (phosphate) and a product analog (vanadate) on the phytase activity. When the concentration of phytate was less than 0.6 mmol/L, the reaction rate increased with the increasing concentration of phytate. And when it was between 0.6 mmol/L and 1.6 mmol/L, the reaction rate changed little, but when it was more than 1.6 mmol/L, the reaction rate decreased with the increasing concentration of phytate. The result showed that high concentrations of phytate inhibited the phytase activity. The K’s value for phytate was estimated to be 2.0 mmol/L. The enzyme activity was inhibited by vanadate with phytate and pNPP as substrate, respectively.The inhibition type was uncompetitive, with the Ki values 62.89 μmol/L and2.42 μmol/L, respectively. The enzyme activity was also inhibited by phosphate with pNPP as substrate and the inhibition type was competitive with the Ki 1.45 mmol/L.更多还原