葡萄汁有孢汉逊酵母β-葡萄糖苷酶的提取与纯化

The Extraction and Purification of β-Glucosidase from Hanseniaspora uvarum Strain

研究葡萄汁有孢汉逊酵母β-葡萄糖苷酶的提取纯化步骤,为其增香酿造特性的研究提供技术支持。首先将优选酵母细胞在振幅为80的超声波下破碎20min,进行β-葡萄糖苷酶的酶活定位,然后对优选菌株的培养条件和糖苷酶的分离纯化条件进行优化。结果表明,供试酵母菌株的β-葡萄糖苷酶为胞外酶,其最佳培养及提取纯化工艺:酵母在28℃发酵培养基中振荡培养72h,4℃10000r/min离心10min,收集上清液,然后经80%硫酸铵盐析、DEAE-Sepharose-FF阴离子交换层析与SephacrylS-200凝胶过滤层析3步纯化,得到β-葡萄糖苷酶液。纯化后β-葡萄糖苷酶回收率为6.91%,比酶活由0.081U/mg提高至1.998U/mg,为纯化前的24.56倍,纯化效果明显。

To provide the theoretical and technical guidance for its application in the winemaking of aroma enhancement, the extraction and purification of β-glucosidase from one selected Hanseniaspora uvarum strain were studied. In the experiment, the isolate cells were broken under ultrasonic(80 amplitude) for 20 min to locate the β-glucosidase firstly. Then, the yeast cultivation conditions, the extraction and purification steps of β-glucosidase were explored and optimized.Results showed that the β-glucosidase from Hanseniaspora uvarum is extracellular enzyme, its best cultivation and extraction conditions for the crude enzyme were cultivated in fermentation medium in 28 ℃ for 72 h, and 10 000 r/min centrifuged in 4 ℃ for 10 min, collecting supernatant. Then the crude β-glucosidase was purified by 80% ammonium sulfate precipitation, DEAE-Sepharose-FF anion exchange chromatography and Sephacryl S-200 gel chromatography. After purification, the recovery of the β-glucosidase was 6.91% and the enzyme activity increased from 0.081 U/mg to 1.998 U/mg (24.56 times improved), purification effect is obvious.