重组内切纤维素酶Fpendo5A转化三七总皂苷

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.

Transformation of Total Notoginsenosides by Recombinant Endocellulase Fpendo5A

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 spec-trometry(RRLC / Q-TOF-MS) was performed to investigate the biotransformation process of the total notogin-senosides 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 acidophi-lic endocellulase. Also, Fpendo5A showed high biotransformation ability for ginsenoside Ra1 , Rb1 , Rc, Rd and Rg3 , which are the main components in the total notoginsenosides. Ra1 , Rb1 , Rc, Rd and Rg3 were con-verted to Rb2 , Gyp ⅩⅦ, CMC1 , F2 and Rh2 , respectively by Fpendo5A.