重组内切纤维素酶Fpendo5A转化三七总皂苷

doi:10.7503/cjcu20170314

摘要/Abstract

摘要：

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Ra₁, Rb₁, Rc, Rd和Rg₃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Ra₁, Rb₁, Rc, Rd和Rg₃的转化产物分别为Rb₂, Gyp ⅩⅦ, CMC₁, F₂和Rh₂. 由此可见, Fpendo5A通过水解Rb₁, Rc, Rd和Rg₃的C3位的β-(1,2)糖苷键分别生成Gyp ⅩⅦ, CMC₁, F₂和Rh₂. 在转化Ra₁时, Fpendo5A通过水解Ra₁的C20位的α-(1,4)木糖苷键生成Rb₂.

关键词: 三七皂苷, 内切纤维素酶, 生物转化, 液相色谱-质谱联用

Abstract:

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 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 converted to Rb₂, Gyp ⅩⅦ, CMC₁, F₂ and Rh₂, respectively by Fpendo5A.

Key words: Notoginsenoside, Endocellulase, Biotransformation, High performance liquid chromatography-mass spectrometry(HPLC-MS)

中图分类号:

O629
Q556

TrendMD:

李男, 赵幻希, 李晶, 王楠, 于渤浩, 越皓, 于珊珊. 重组内切纤维素酶Fpendo5A转化三七总皂苷. 高等学校化学学报, 2017, 38(12): 2185.

LI Nan, ZHAO Huanxi, LI Jing, WANG Nan, YU Bohao, YUE Hao, YU Shanshan. Transformation of Total Notoginsenosides by Recombinant Endocellulase Fpendo5A^†. Chem. J. Chinese Universities, 2017, 38(12): 2185.

图/表 5

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Peak No.	Compd.	t_R/min	Molecular formula	MS(calcd. mass), m/z	10⁶ Error	MS/MS fragment ions of [M-H]^-(mass accuracy < 5×10^-6)
1	Ra₁	21.74	C₅₈H₉₈O₂₆	1209.6242 (1209.6274)	2.7	1077.5839[M-H-Xyl ]^-, 945.5418[M-H-Xyl-Ara(p)]^-, 783.4874[M-H-Xyl-Ara-Glu]^-, 621.4336[M-H-Xyl-Ara-GlcGlc]^-, 459.5418[M-H-Xyl-Ara-GlcGlcGlc]^-, 323.0862[GlcGlc-H]^-
2	Rb₁	22.67	C₅₄H₉₂O₂₃	1107.5957 (1107.5976)	1.7	945.5294[M-H-Glc]^-, 783.4787[M-H-GlcGlc]^-,621.4295[M-H-GlcGlcGlc]^-, 459.33790[M-H-GlcGlcGlcGlc]^-, 323.0832[GlcGlc-H]^-
3	Rc	24.01	C₅₃H₉₀O₂₂	1077.5851 (1077.5875)	2.2	945.5193[M-H-Ara(f)]^-, 783.4760[M-H-Ara-Glc]^-, 621.4169[M-H-Ara-GlcGlc]^-, 459.3660[M-H-Ara-GlcGlcGlc]^-
4	Rb₂	24.93	C₅₃H₉₀O₂₂	1077.5851 (1077.5812)	3.6	945.5348[M-H-Ara(p)]^-, 783.4898[M-H-Ara-Glc]^-, 621.4365[M-H-Ara-GlcGlc]^-, 459.3852[M-H-Ara-GlcGlcGlc]^-
5	Rd	25.76	C₄₈H₈₂O₁₈	945.5428 (945.5462)	3.6	783.7832[M-H-Glc]^-, 621.5318[M-H-GlcGlc]^-, 459.5767[M-H-GlcGlcGlc]^-, 323.3125[GlcGlc-H]^-
6	Gyp ⅩⅦ	26.57	C₄₈H₈₂O₁₈	945.5428 (945.5437)	0.95	783.4615[M-H-Glc]^-, 621.4111[M-H-GlcGlc]^-, 459.9876[M-H-GlcGlcGlc]^-
7	CMC₁	28.28	C₄₇H₈₀O₁₇	915.5323 (915.5348)	2.7	783.1984[M-H-Ara(f)]^-, 621.6952[M-H-Ara-Glc]^-, 459.4832[M-H-Ara-GlcGlc]^-
8	F₂	29.11	C₄₂H₇₂O₁₃	783.4900 (783.4870)	3.8	621.3217[M-H-Glc]^-, 459.1521[M-H-GlcGlc]^-
9	Rg₃	30.83	C₄₂H₇₂O₁₃	783.4900 (783.4933)	4.2	621.4223[M-H-Glc]^-, 459.3791[M-H-GlcGlc]^-
10	Rh₂	33.66	C₃₆H₆₂O₈	621.4372 (621.4347)	4.0	459.3563[M-H-Glc]^-

Peak No.	Compd.	t_R/min	Molecular formula	MS(calcd. mass), m/z	10⁶ Error	MS/MS fragment ions of [M-H]^-(mass accuracy < 5×10^-6)
1	Ra₁	21.74	C₅₈H₉₈O₂₆	1209.6242 (1209.6274)	2.7	1077.5839[M-H-Xyl ]^-, 945.5418[M-H-Xyl-Ara(p)]^-, 783.4874[M-H-Xyl-Ara-Glu]^-, 621.4336[M-H-Xyl-Ara-GlcGlc]^-, 459.5418[M-H-Xyl-Ara-GlcGlcGlc]^-, 323.0862[GlcGlc-H]^-
2	Rb₁	22.67	C₅₄H₉₂O₂₃	1107.5957 (1107.5976)	1.7	945.5294[M-H-Glc]^-, 783.4787[M-H-GlcGlc]^-,621.4295[M-H-GlcGlcGlc]^-, 459.33790[M-H-GlcGlcGlcGlc]^-, 323.0832[GlcGlc-H]^-
3	Rc	24.01	C₅₃H₉₀O₂₂	1077.5851 (1077.5875)	2.2	945.5193[M-H-Ara(f)]^-, 783.4760[M-H-Ara-Glc]^-, 621.4169[M-H-Ara-GlcGlc]^-, 459.3660[M-H-Ara-GlcGlcGlc]^-
4	Rb₂	24.93	C₅₃H₉₀O₂₂	1077.5851 (1077.5812)	3.6	945.5348[M-H-Ara(p)]^-, 783.4898[M-H-Ara-Glc]^-, 621.4365[M-H-Ara-GlcGlc]^-, 459.3852[M-H-Ara-GlcGlcGlc]^-
5	Rd	25.76	C₄₈H₈₂O₁₈	945.5428 (945.5462)	3.6	783.7832[M-H-Glc]^-, 621.5318[M-H-GlcGlc]^-, 459.5767[M-H-GlcGlcGlc]^-, 323.3125[GlcGlc-H]^-
6	Gyp ⅩⅦ	26.57	C₄₈H₈₂O₁₈	945.5428 (945.5437)	0.95	783.4615[M-H-Glc]^-, 621.4111[M-H-GlcGlc]^-, 459.9876[M-H-GlcGlcGlc]^-
7	CMC₁	28.28	C₄₇H₈₀O₁₇	915.5323 (915.5348)	2.7	783.1984[M-H-Ara(f)]^-, 621.6952[M-H-Ara-Glc]^-, 459.4832[M-H-Ara-GlcGlc]^-
8	F₂	29.11	C₄₂H₇₂O₁₃	783.4900 (783.4870)	3.8	621.3217[M-H-Glc]^-, 459.1521[M-H-GlcGlc]^-
9	Rg₃	30.83	C₄₂H₇₂O₁₃	783.4900 (783.4933)	4.2	621.4223[M-H-Glc]^-, 459.3791[M-H-GlcGlc]^-
10	Rh₂	33.66	C₃₆H₆₂O₈	621.4372 (621.4347)	4.0	459.3563[M-H-Glc]^-