不同添加剂对芋豆蛋白a修饰二氧化钛纳米复合材料固定化β-半乳糖苷酶结构和活性的影响

A. Shafi, Q. Husain
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引用次数: 1

摘要

解释β-半乳糖苷酶的活性和结构之间的关系,是了解酶的构象对其催化作用的影响的必要条件。本研究全面解释了乙二胺四乙酸(EDTA)、十二烷基硫酸钠(SDS)、二硫苏糖醇(DTT)和尿素等添加剂对β-半乳糖苷酶活性和结构的影响。测定了β-半乳糖苷酶在不同离子强度和温度下随时间变化的活性。利用紫外可见光谱和本征荧光光谱对添加剂存在下酶的二级和三级结构的变化进行了结构研究。固定化酶在不同环境条件下均表现出较好的稳定性。活性分析表明,在SDS和尿素的存在下,β-半乳糖苷酶的失活呈浓度依赖性,这表明在活性位点附近存在疏水和带电残基。在EDTA存在的情况下,活性下降,证实了β-半乳糖苷酶是一种金属酶。DTT存在时酶活性的增强表明在催化中心附近存在半胱氨酸残基。在紫外可见光谱和本征荧光光谱研究中,天然酶在DTT、SDS和尿素存在下表现出明显的构象转变,而在EDTA存在下几乎没有变化。然而,固定化酶可以抵抗显著的结构变化。总之,本研究提供了β-半乳糖苷酶活性与构象稳定性之间关系的详细描述。
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Effect of Different Additives on the Structure and Activity of β-Galactosidase Immobilized on a Concanavalin A–Modified Silica-Coated Titanium Dioxide Nanocomposite
Interpreting the relationship between the activity and structure of β-galactosidase is necessary to perceive the impact of the enzyme’s conformation on its catalysis. The current study thoroughly explains the effects of additives such as ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), dithiothreitol (DTT), and urea on β-galactosidase activity and structure. β-Galactosidase activity was determined at various ionic strengths and temperatures as a function of time. Structural studies evaluating changes in the secondary and tertiary structures of the enzyme in the presence of the additives were conducted using ultraviolet (UV)-visible and intrinsic fluorescence spectroscopy. The immobilized enzyme showed enhanced stability under different environmental conditions. Activity assays demonstrated concentration-dependent inactivation of β-galactosidase in the presence of SDS and urea, which suggests that hydrophobic and charged residues are present near the active site. In the presence of EDTA, loss in activity was noted, which confirms that β-galactosidase is a metalloenzyme. Enhancement in enzyme activity in the presence of DTT suggests the presence of a cysteine residue near the catalytic center. In UV-visible and intrinsic fluorescence spectroscopy studies, the native enzyme showed significant conformational transitions in the presence of DTT, SDS, and urea and very few changes in the presence of EDTA. However, the immobilized enzyme could resist significant structural changes. In conclusion, this study provides a detailed description of the association between the activity and conformational stability of β-galactosidase.
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