嗜酸热脂环酸杆菌中甘露聚糖酶活性位点的确立  

Identification of the catalytic residues of mannanase from Alicyclobacillus acidocaldarius

作　　者：徐书景[1] 张彩凤[2] 薛张伟[2] 何广正[2] 鞠建松[2] 赵宝华[2] 

机构地区：[1]河北师范大学旅游学院,石家庄050016 [2]河北师范大学生命科学学院,石家庄050016

出　　处：《微生物学报》2011年第1期66-74,共9页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30970628);河北省留学回国人员资助项目(20100705);河北师范大学博士启动基金项目(L2009B13)~~

摘　　要：【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。[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 analyzed by thin layer chromatography （TLC） and the dinitrosalicylic acid （DNS） methods for enzyme assay.[Results]Seven mutants,E151A,E159A,E231A,C150A,E151Q,E231Q and double mutation E151QE231Q were successful constructed.Mutant E159A showed similar activities with wild type,and C150A mutation resulted in only a 3-fold reduction in the activities,but mutations E151A,E231A,E151Q,E231Q and E151QE231Q resulted in sharp decreases or loss in the activities,indicating that Glu151 and Glu231 play critical roles in AaManA activity.Furthermore,the presence of Glu151 at the C terminus of β4 and Glu231 at the C terminus of β7 was entirely consistent with the positions of the acid /base catalyst and the nucleophile catalyst of a GH-A enzyme,respectively.[Conclusion]By combining the results of TLC and enzyme assay of those mutants and the structural comparisons,it was confirmed that Glu151 and Glu231 fulfilled the roles of an acid /base catalyst and nucleophile catalyst in AaManA,respectively.

关 键 词：甘露聚糖酶 定点突变 活性位点 

分 类 号：Q93[生物学—微生物学]