对硝基酚-α-D-吡喃半乳糖法测定饲用α-半乳糖苷酶(黑曲霉)活力的方法  

Determination of α-Galactosidase ( Aspergillus niger) Used in Feed with p -Nitrophenyl-α-D-Galactospyranoside

作　　者：潘宝海[1] 李德发[1] 陆文清[1] 代建国[1] 

机构地区：[1]中国农业大学动物科技学院,北京100094

出　　处：《中国农业大学学报》2002年第5期107-111,共5页Journal of China Agricultural University

基　　金：国家自然科学基金资助项目 (39770 5 49)

摘　　要：旨在建立一种适合于测定饲用α-半乳糖苷酶活力的检测方法。实验利用对硝基酚 -α- D-吡喃半乳糖(p- NPG)作为酶反应底物 ,通过 4 0 0 nm比色检测酶反应所释放的对硝基酚的量来测定饲用酶制剂中 α-半乳糖苷酶的活性。研究表明 ,反应液中所含的对硝基酚的量大于 15 μmol.m L-1时 ,吸光值超过仪器的检测范围 ;酶制剂稀释倍数对对硝基酚比色法的影响较小 ,本底占总酶活的 10 %～ 16 % ,当酶稀释 10 0倍时 ,本底对酶活的影响最小 ;在测定酶活反应时间为 6 min时 ,已经有约 10 %的对硝基酚 - α- D-吡喃半乳糖水解 ,当酶液的酶活在 2 .0 U.m L-1以内 ,测试结果较为准确。考虑到饲用酶制剂的作用环境 ,建议反应温度为 37℃、反应体系 p H 5 .0。This experiment was conducted to determine the α galactosidase activity produced by Aspergillus niger , which was used as an animal feed additive. p Nitrophenyl α D galactospyranoside ( p NPG) was used as substrate to measure the amount of p nitrophenol released in the enzyme reaction with spectrometer at 400nm. It was found that if the amount of p nitrophenol was higher than 15μmol·mL -1 , OD was beyond the limitation of measurement. The dilution rate had little effect on the enzyme activity with p NPG as substrate, accounting about 10%～16% of the total enzyme activity. When the dilution rate is 100, the effect of substrate on the enzyme activity was lowest. The reaction time should be controlled for 6 min when about 10 percent of p NPG was hydrolyzed. The enzyme activity in the dilution should be lower than 2.0 UmL -1 for the sake of accuracy. It was showed that the temperature and pH of reaction solution should be controlled at 37℃ and 5.0 respectively, considering the condition of animal digestive tracts. In conclusion, using p NPG as a substrate to determine feed α-galactosidase is a practical and accurate method.

关 键 词：对硝基酚-α-D-吡喃半乳糖法 测定 Α-半乳糖苷酶 黑曲霉 饲用酶制剂 检测方法 酶活力 

分 类 号：S816.79[农业科学—饲料科学] TQ925.9[农业科学—畜牧学]