| 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。 |
| 关键词: TTC还原法 脱氢酶 真菌 粗酶制剂 |
| DOI： |
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| 基金项目:陕西博秦生物工程有限公司微生物增油项目 |
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| Optimization of determination method for dehydrogenase activity of crude enzyme preparation from fungi |
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MA Xin,GAO Hui,ZHANG Jun-hui,et al
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| Abstract: |
| 【Objective】The study explored the influencing factors on dehydrogenase activity of enzyme preparation from fungi to determine the test method.【Method】Triphenyltetrazolium chloride (TTC)-reduction method was used to assay the dehydrogenase activity of fungal crude enzyme and analyze the effect of mass ratio of crude enzyme preparation to deionized water,activation time,activation temperature, oscillation rate,stirring time,solid-liquid separation method,activation agent,filter medium,and chromogenic time on dehydrogenase activity.【Result】①Activation,separation method and color reaction time had significant influence on dehydrogenase activity.②The measurement method of dehydrogenase activity was determined.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/min 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 CH3COOC2H5 was added.Finally,it was put into 4 ℃ refrigerator for 5-6 h before the clear organic layer was assayed by colorimetry at wavelength of 485 nm and dehydrogenase activity was calculated by comparing to standard curve.【Conclusion】Determination method of dehydrogenase activity of crude enzyme preparation from fungi was optimized. |
| Key words: TTC-reduction method dehydrogenase fungi crude enzyme preparation |
