HPLC-MSn法检测毛白杨CCR酶活及反应产物4-羟基肉桂醛类的制备  

Research of 4-hydroxycinnamaldehydes-production of CCR enzyme reaction

作　　者：侯佳音[1] 刘淑欣[1] 张瀚文[1] 蒋湘宁[1] 陈雪梅[1] 

机构地区：[1]北京林业大学生物科学与技术学院,北京100083

出　　处：《河北农业大学学报》2015年第2期25-30,共6页Journal of Hebei Agricultural University

基　　金：国家基础科学人才培养基金资助项目(No.J1103516)

摘　　要：为更直观、便捷地研究木质素合成途径中CCR的酶学特性,从产物生成的方向来监测CCR的酶学特性。试题采取化学合成方法制备CCR的产物——4-羟基肉桂醛物质(香豆醛、松柏醛、咖啡醛)并利用HPLC进行纯化,液相色谱质谱联用仪(HPLC-MSn)进行鉴定,合成的4-羟基肉桂醛可作为HPLC-MSn技术研究CCR酶学特性时的标样。建立了一套HPLC-MSn技术检测CCR酶活的新方法,将毛白杨重组CCR蛋白在大肠杆菌中高效表达并利用亲和层析法获得电泳纯CCR蛋白,利用HPLC-MSn技术监测CCR酶对4种底物的酶学特性。结果显示,通过该方法能够监测到CCR底物的减少和产物的增加过程,为木质素合成途径中关键酶的酶学活性研究提供新方法。In order to investigate thoroughly the enzymatic properties of CCR which plays criti- cal role in lignin biosynthesis, we analyzed the catalytic characteristics of CCR from perspec- tive of products formation. Chemical method was used for synthesis of 4 - hydroxycinnamalde- hydes （p-coumaraldehyde, coniferaldehyde, caffeylaldehyde） which are the products of CCR enzymatic reaction. 4 - Hydroxycinnamaldehydes were then purified by HPLC and detected by HPLC- MSn. Results showed that the synthesized 4- hydroxycinnamaldehydes can be used as standards in research of CCR. We also established a new method to detect CCR activity u- sing HPLC - MSn. The recombinant CCR protein was expressed efficiently and purified by af- finity chromatography. The CCR activity assay of the enzymatic reactions with multiple sub- strates was performed using HPLC - MS system. The results showed that the method can ef- fectively monitor the change in both substrate and product, which provides new insights for the study of catalytic characteristics of key enzymes in lignin biosynthesis.

关 键 词：肉桂酰辅酶A还原酶 HPLC-MS 4-羟基肉桂醛类 化学合成 毛白杨 酶活性 

分 类 号：S792.117[农业科学—林木遗传育种]