木聚糖酶的分离纯化是对其进行酶学研究和分子改良研究的基础.利用实验室选育的黑曲霉菌株Aspergillus niger SM24/a进行木聚糖酶发酵,粗酶液经过(NH4)2SO4分级沉淀Bio-Gel P6除盐、UNO sphere Q阴离子交换和Enrich SEC70凝胶色谱层析四个步骤的分离纯化,成功获得了3种木聚糖酶蛋白定义为X-Ⅰ、X-Ⅱ和X-Ⅲ.随着纯化步骤的增加,各组分酶比活力得到显著提高,其数值分别为37.41、34.56和53.96 U/mg,纯化倍数分别为3.96、3.66和5.72.经质谱分析和蛋白氨基酸序列比对,初步认定X-Ⅰ属于糖基水解酶第十家族内切-β-1,4-木聚糖酶,X-Ⅱ和X-Ⅲ均属于糖基水解酶第十一家族木聚糖酶.%Separation and purification of xylanase are the base of carrying out its enzymological study and molecular improvement.Xylanase fermentation was carried out using Aspergillus niger SM24/a bred in the lab.The crude enzyme solution was fractionally precipitated with (NH4)2SO4,desalinated by Bio-Gel P6,separated and purified with UNO sphere Q anion exchange and Enrich SEC70 gel chromatography,these four steps,and successfully obtained three kinds of xylanase,and named as X-Ⅰ,X-Ⅱ,and X-Ⅲ.As the increment of purification steps,the specific activity of each component was significantly improved,their numerical value respectively were 37.41 U/mg,34.56 U/mg and 53.96 U/mg,and the purification folds were 3.96,3.66,and 5.72 respectively.Through mass spectrometry and amino acid sequence comparison,it was initially confirmed that X-Ⅰ belonged to endo-β-1,4-xylanase of glycosyl hydrolase family 10,and both X-Ⅱ and X-Ⅱ belonged to xylanase of glycosyl hydrolase family 11.
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