嗜酸热脂环酸杆菌中甘露聚糖酶活性位点的确立

【目的】通过定点突变确定嗜酸热脂环酸杆菌中甘露聚糖酶的活性催化位点。【方法】根据序列比对和GH53家族的结构信息选择可能的催化活性位点,利用重叠PCR法构建定点突变体,采用薄层层析(TLC)法和3,5-二硝基水杨酸(DNS)法检测各酶蛋白活性。【结果】通过重叠PCR法成功构建了7个位点的突变体,其中第150和159位的氨基酸突变对活性改变甚少或几乎没有,而第151和231位谷氨酸的羧基基团的改变以及双位点突变体E2Q则导致其对各种底物催化活性的丧失,说明位于β4和β7折叠的C末端的E151和E231的羧基基团作为功能基团参与了催化反应。【结论】E151和E231分别是新型甘露聚糖酶AaManA的酸碱催化位点和亲核催化位点。

Identification of the catalytic residues of mannanase from Alicyclobacillus acidocaldarius

Objective]To identify the catalytic residues of mannanase AaManA from Alicyclobacillus acidocaldarius.Methods]Based on the sequence alignment by ClustalX and ESPript and the structure information of GH-53 family,the possible catalytic residues were selected and mutated by overlap extension PCR.The protein of wild type and mutant were expressed in E.coli BL21 (DE3) and ordinal purified by Ni-NTA affinity chromatography,gel-filtrate chromatography and ion-exchange chromatography.The purified protein was ana...