紫外分光光度法测定啤酒大麦籽粒脂氧合酶活力的研究

Determination of Lipoxygenase Activity in Barley Grain by UV Spectrophotometry

为了明确影响啤酒大麦籽粒脂氧合酶(LOX-1)活力的参数,以甘啤4号啤酒大麦籽粒为研究对象,采用紫外分光光度法探究底物浓度、提取缓冲液p H、反应体系缓冲液p H、提取时间、粗酶加入量∶底物加入量等对LOX-1活力的影响。结果表明,LOX-1活力随底物浓度的增加表现为先增加后减小的趋势,当底物浓度达到0.30mmol·L^(-1)时,LOX-1活力显著高于其它处理;随提取缓冲液p H的增加,LOX-1活力表现为双峰变化趋势,且在p H值5.0处,酶活力为9.85U·g^(-1),显著高于其它处理;粗酶提取时间为30min时,LOX-1活力显著高于其它处理,低于或者高于30min,酶活力均呈现降低趋势;LOX-1活力随反应体系p H的增加,表现为双峰变化趋势,当p H值为6.4时,酶活力最大,且与其它处理间存在显著差异;在其它条件不变,粗酶加入量为50μL的情况下,增加底物加入量,LOX-1活力表现为先增加后减小的趋势,且底物加入量为200μL时,酶活力显著高于其它处理。最终确定紫外分光光度法的测定参数:粗酶提取时间为30min,底物浓度为0.25mmol·L^(-1),提取液和反应体系缓冲液分别为p H值5.0醋酸盐缓冲液与p H值6.4磷酸盐缓冲液,粗酶加入酶量∶底物加入量为1∶4。研究结果为紫外分光光度法在啤酒大麦籽粒LOX-1活力测定中的应用提供了参考。

Barley grains of Ganpi 4 were selected as the research object to determine the parameters of UV spectrophotometry for the lipoxygenase（ LOX- 1） activity measurement. The effects of substrate concentration,extraction buffer p H,buffer p H of reaction system,extraction time and the ratio between extraction and substrate addition on LOX-1 activity measured by UV spectrophotometry were studied. The results indicated that LOX- 1 activity increased at first,and then decreased with the substrate concentration increasing,and the peak activity appeared at 0. 30mmol·L-（-1）of substrate concentration,which was significantly higher than that of the other treatments; with the increasing of extraction buffer p H,the LOX- 1 activity varied with two peaks and the enzyme activity reached 9. 85 U·g-（-1）at p H5. 0,which was significantly higher than that of the other treatments; LOX- 1 activity which was extracted for 30 min was significantly higher than that of the other treatments,and the enzyme activity had the trend of decrease at the extraction time which above or below 30min; when the extraction addition was 50 μL and other conditions remained unchanged,LOX-1 activity increased at first,and then decreased as the substrate addition increased,and the enzyme activity with the substrate addition of 200 μL was significantly higher than that the other treatments. Finally,the parameters of UV spectrophotometry were determined： 30 minutes extraction time, 0. 25 mmol ·L-（-1）substrate concentration,p H 5. 0 acetate extraction buffer,p H 6. 4 phosphate reaction system buffer,and 1∶ 4 as the ratio between extraction and substrate addition. The results can be used as the reference for the determination of LOX- 1 activity in barley grain by UV spectrophotometry.