黑曲霉产木聚糖酶的分离纯化与鉴定研究

Isolation,Purification,and Identification of Xylanase from Aspergillus niger SM24/a

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(NH_4)_2SO_4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.