黑曲霉DB056分泌的柚苷酶分离纯化及酶学性质研究

Study on Purification and Enzymalogy Characteristic of Naringinase from Aspergillus Niger DB056

对黑曲霉DB056发酵产生的柚苷酶进行分离纯化并研究其基本特性。粗酶液经硫酸铵沉淀、离子交换层析和凝胶过滤层析分离,所得柚苷酶的比活力为1716.41U/mg蛋白,纯化倍数为17.52倍。柚苷酶具有4个相对分子质量的组分,较适pH值范围4.0～5.0,最稳定的pH4.5;该酶适宜的作用温度范围45～55℃,温度50℃时酶活力最大,45℃和50℃条件下可以分别稳定4h和2h,55℃时柚苷酶1h失活50%;Ag+、Hg2+对柚苷酶具有强烈的抑制作用,其它金属离子对柚苷酶的影响随离子浓度的不同而不同,如0.1mmol/LCa2+、K+、Al3+、Mn2+、Cu2+可提高柚苷酶的活力,而5.0mmol/LFe2+、Al3+对柚苷酶显示抑制作用。

Naringinase produced by Aspergillus niger DB056 were isolated and purified and its basic characteristics were studied.The naringinase was obtained by ammonium sulphate precipitation,ion exchange and gel filtration chromatography with an overall recovery of 4.30%,a specific activity of 1716.41 U/mg of protein and a purifying fold of 17.52.The enzyme had an optimum pH of 4.0～5.0 and temperature of 45～50 ℃,respectively,at the same time,it was stable in pH 4.5.The naringinase was stable at 45 ℃ for 4 h,and at 50 ℃ for 2 h whereas at 55 ℃ the enzyme showed 50% inactivation after 1 h of incubation.Ca2＋、K＋、Al3＋、Mn2＋、Cu2＋ and so on could improve the enzyme activity at a concentration of 0.1 mmol/L,whereas,Fe2＋、 Al3＋ inhibited the enzyme activity at a concentration of 5.0 mmol/L,and Ag＋、 Hg2＋ strongly inhibited the enzyme activity.