摘要
【目的】探究酶液制备及测定条件对真菌粗酶制剂脱氢酶活性的影响，确定真菌粗酶制剂脱氢酶活性测定方法。【方法】采用氯化三苯基四氮唑（TTC）还原法测定真菌粗酶制剂的脱氢酶活性，分析粗酶制剂与去离子水质量比、活化时间、活化温度、振荡速率、搅拌时间、固液分离方法、活化剂种类、过滤介质及显色时间对脱氢酶活性的影响。【结果】①真菌粗酶制剂活化、分离方式及显色时间对真菌粗酶制剂脱氢酶活性测定有明显影响。②确定真菌粗酶制剂脱氢酶活性测定优化方法为：将粗酶制剂与去离子水按照质量比1∶40装入三角瓶，在28 ℃、120 r/min恒温摇床中振荡15 h，用快速滤纸过滤获得酶液；将酶液与TTC葡萄糖溶液混匀，在40 ℃恒温水浴锅中反应显色，待反应液出现红色后计时，维持90 min完成显色反应，取出后加乙酸乙酯充分摇匀，4 ℃冰箱中静置5～6 h，取上层澄清有机相在波长485 nm比色，根据标准曲线计算脱氢酶活性。【结论】获得了测定真菌粗酶制剂脱氢酶活性的测定方法。
[Objective] The study explored the influencing factors on dehydrogenase activity of enzyme preparation from fungi to determine the test method. [Method] Triphenyltetrazolium chloride （TTC）-re- duetion method was used to assay the dehydrogenase activity of fungal crude enzyme and analyze the effect of mass ratio of erude enzyme preparation to deionized water,activation time,activation temperature, oscil- lation rate, stirring time, solid-liquid separation method, activation agent, filter medium, and ehromogenie time on dehydrogenase activity. [Result] C）Aetivation, separation method and color reaction time had sig- nificant influence on dehydrogenase activity. （1）The measurement method of dehydrogenase activity was de- termined. Crude enzyme and deionized water were mixed at the mass ratio of 1 ： 40 in the triangle bottle before being shaken with oscillation rate of 120 r/rain at 28 ℃ for 15 h. Then rapid filter paper was used to separate mixture and enzyme solution and TTC-glucose reacted in thermostatic water bath at 40 ℃. As the reaction mixture became reddish,it was maintained for 90 min before 5 mL CH3COOC2 H5 was added. Fi- nally,it was put into 4 ℃ refrigerator for 5--6 h before the clear organic layer was assayed by eolorimetry at wavelength of 485 nm and dehydrogenase activity was calculated by comparing to standard curve. [Con-clusion] Determination method of dehydrogenase activity of crude enzyme preparation from fungi was optimized.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2016年第1期155-161,176,共8页
Journal of Northwest A&F University(Natural Science Edition)
基金
陕西博秦生物工程有限公司微生物增油项目
关键词
TTC还原法
脱氢酶
真菌
粗酶制剂
TTC-reduction method
dehydrogenase
fungi
crude enzyme preparation
