黑芥子酶提取及活性测定方法的改进  

An improved method for extracting and activity determining of myrosinase

作　　者：张蕾[1] 庞秋颖[1] 王洋[1] 

机构地区：[1]东北林业大学盐碱地生物资源环境研究中心,东北油田盐碱植被恢复与重建教育部重点实验室,黑龙江哈尔滨150040

出　　处：《东北师大学报（自然科学版）》2011年第1期118-121,共4页Journal of Northeast Normal University(Natural Science Edition)

基　　金：国家自然科学基金海外青年学者合作研究基金资助项目(30528013);国家自然科学基金资助项目(30670325);东北林业大学研究生论文资助项目(gram09);新世纪优秀人才支持计划项目(NCET-05-0328)

摘　　要：以拟南芥为材料,对以往黑芥子酶的提取方法和活性测定方法进行了改进,通过实验优选出最佳的提取和活性测定条件.改进后的方法提取样品量只有150mg,提取缓冲溶液1mL,离心回收上清作为酶液;3mL反应体系溶液(含0.2mmol/L黑芥子硫苷酸钾)置于1cm光路石英比色杯中,37℃条件下平衡后,向反应体系溶液中添加150μL酶液,预反应3min,而后在227nm波长下测量吸光度5min(反应时间),计算酶活.改进后的方法取样量小,操作较为简便,适于较多样品的平行测定.An improved method of extraction and activity determination for the myrosinase were investigated and the best method was proposed as follows：150 mg sample of tissue was solubilized in 1 mL of extraction buffer.The sample was centrifuged then the supernatant was collected and used as myrosinase extract.3 mL reaction mixture which contains 0.2 mmol/L sinigrin was allowed to equilibrate at 37℃ then the reaction was initiated by adding 150 μL enzyme extract to the 1cm cuvette.Decline in optical density as a result of sinigrin breakdown was plotted at 227 nm for 5 min after a 3 min pre-reaction period using a spectrophotometer.The improved method was performed by using less plant sample quantity and easy to operate,which is suitable for parallel determination of many samples.

关 键 词：拟南芥 黑芥子酶 测定方法 

分 类 号：Q946.5[生物学—植物学]