[关键词]
[摘要]
为寻求高效、安全、快捷地运用高压脉冲电场(PEF)对酶进行活化改性的可行性。本项目以葡萄糖淀粉酶为对象,研究PEF处理对其活性、酶学性质、动力学参数的影响,并借助SDS-PAGE、荧光光谱和圆二色谱(CD)等分析其构象变化。研究结果表明:当电场强度为15 kV/cm,流速为80 mL/min,葡萄糖淀粉酶活性激活最为明显,与原酶液相比增加20.74±0.71%,且该激活效应在4 ℃环境下能维持17 h;并且PEF处理能拓宽葡萄糖淀粉酶的最适反应温度范围,降低Km,增大Vmax,提高其催化效率,但最适pH范围几乎不变。同时SDS-PAGE结果表明PEF处理不改变葡萄糖淀粉酶分子量,其一级结构不变;圆二色光谱结果表明,PEF处理使其α-螺旋、β-折叠含量分别增加3.00%和2.80%,酶空间结构趋于有序化,二级结构发生改变;而内源荧光分析显示PEF处理使酶吸收峰增强,Trp残基等微环境暴露,三级结构发生变化。
[Key word]
[Abstract]
The efficient, safe, and rapid application of pulsed electric field (PEF) technology to enzymatic modification was studied. The effect of PEF treatment on the enzymatic activity, properties, and kinetic parameters of glucoamylase were investigated, while conformational transitions were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD) and fluorescence spectroscopy. The results showed that the maximum increase in activity obtained was 20.74 ± 0.71% as compared to control, observed when 15 kV/cm electric field intensity and 80 mL/min flow rate were applied and this activation effect lasted for up to 17 h at 4 ℃. Moreover, PEF treatment widened the range of optimum temperature for glucoamylase activity, increased the Vmax and decreased the Km, but had a negligible effect on the optimum pH of glucoamylase. Results of SDS-PAGE indicated that the primary structure of glucoamylase did not change after PEF treatment. However, according to CD and ?uorescence spectra, PEF treatment enhanced the α-helix and β-sheet content in glucoamylase by 3.00% and 2.80%, respectively, leading to more ordered conformation. Additionally, intrinsic fluorescence spectra analysis showed that PEF treatment resulted in a red shift of the emission maximum of glucoamylase due to the exposed microenvironment of Trp residue. Therefore, based on these results, both, secondary and tertiary structures of glucoamylase were modified.
[中图分类号]
[基金项目]
重庆市自然科学基金项目(cstc2012jjA80045)