本文针对黑曲霉液态发酵所产柚苷酶的分离纯化工艺及其酶学性质进行研究。将黑曲霉液态发酵所产酶液，依次通过30%~70%硫酸铵盐析、膜透析和DEAE-Sepharose FF阴离子交换层析后得到高纯度柚苷酶。经SDS-PAGE凝胶电泳仅得到一条清晰条带，分子量约为65.10 ku。纯化获得的柚苷酶比酶活可达6932.54 U/mg，纯化倍数和酶活回收率分别为13.82倍和13.30%，并且能有效水解柚皮苷。进一步研究发现，该酶的最适反应pH为4.5，最适作用温度为50 ℃，在20 ℃~50 ℃及pH 3.0~6.0范围内有良好的稳定性。在一定浓度范围内，K+、Ca2+和Na+对柚苷酶活性有促进作用，而Fe3+、SDS和EDTA-Na2对其活性有明显的抑制作用。本研究为深入理解柚苷酶分离纯化过程，明确其酶学性质，进一步探索柚苷酶应用于天然活性产物生物转化过程的相关机制提供了基础数据，具有非常重要的科学意义和潜在的应用价值。

The isolation, purification, and enzymatic properties of naringinase produced by liquid fermentation with Aspergillus niger FFCC 848 were investigated. The fermentation broth produced from liquid fermentation by A. niger FFCC 848 was purified by ammonium sulfate fractional precipitation, dialysis, and DEAE-Sepharose FF anion exchange chromatography to yield highly purified naringinase. Only one clear band was observed upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, with a molecular weight of 65.10 ku. The final, purified naringinase showed 13.82-fold purification, with an enzyme activity recovery of 13.30% and specific activity of 6932.54 U/mg, and the enzyme could effectively hydrolyze naringin. Furthermore, the activity of naringinase was stable at 20–50?C and pH 3.0–6.0; optimum activity was observed at 50?C and pH 4.5. In a specific concentration range, the enzyme activity was enhanced by K+, Ca2+, and Na +, but was strongly inhibited by Fe3+, SDS, and EDTA-Na2. This study provides a basis for understanding the purification of naringinase and for further studies of the related mechanism underlying the bioconversion of bioactive natural products with naringinase.

辽宁省自然科学基金（2013020167）；辽宁省教育厅科学研究一般项目（L2015045）；大连工业大学青年基金（67007908）