重组内切纤维素酶Fpendo5A转化三七总皂苷

Transformation of Total Notoginsenosides by Recombinant Endocellulase Fpendo5A

从嗜热细菌基因组中克隆到1个新的纤维素酶基因,并在大肠杆菌中进行了高效可溶性表达,粗酶液经镍柱亲和层析进行分离纯化.利用快速分离液相色谱-四极杆飞行时间质谱联用仪(RRLC/Q-TOF-MS)对重组内切纤维素酶Fpendo5A转化三七总皂苷的产物结构进行了鉴定,并进一步阐明其转化机制.结果表明,该酶的最适反应温度和pH值分别为80℃和5.5.Fpendo5A能够催化三七总皂苷中的主要皂苷成分,即Ra_1,Rb_1,Rc,Rd和Rg_3的侧链糖基的水解反应,但对于不同的皂苷底物,Fpendo5A选择性催化的侧链糖基类型不同.经鉴定,Fpendo5A转化Ra_1,Rb_1,Rc,Rd和Rg_3的转化产物分别为Rb_2,GypⅩⅦ,CMC_1,F_2和Rh_2.由此可见,Fpendo5A通过水解Rb_1,Rc,Rd和Rg3的C3位的β-(1,2)糖苷键分别生成GypⅩⅦ,CMC1,F2和Rh2.在转化Ra_1时,Fpendo5A通过水解Ra_1的C20位的α-(1,4)木糖苷键生成Rb2.

A novel endocellulase（Fpendo5A） was cloned from Fervidobacterium pennivorans DSM9078.After being overexpressed and purified from Escherichia coli,the enzymatic properties of Fpendo5A were investigated.Also,rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry（RRLC/Q-TOF-MS） was performed to investigate the biotransformation process of the total notoginsenosides by Fpendo5A.Fpendo5A exhibited an optimal activity at 80 ℃ and pH=5.5 and showed the highest activity for carboxymethyl cellulose（CMC）,which indicated that Fpendo5A was a thermophilic and acidophilic endocellulase.Also,Fpendo5A showed high biotransformation ability for ginsenoside Ra_1,Rb_1,Rc,Rd and Rg3,which are the main components in the total notoginsenosides.Ra_1,Rb_1,Rc,Rd and Rg_3 were converted to Rb_2,Gyp ⅩⅦ,CMC_1,F_2 and Rh_2,respectively by Fpendo5A.