重组β-木糖苷酶转化人参皂苷Rb3及C-Mx

doi:10.7503/cjcu20190626

摘要/Abstract

摘要：

从Aspergillus niger NG1306菌株中扩增得到β-木糖苷酶基因anxyl, 与pPICZαA连接构建了表达载体pPICZαA-anxyl, 进一步在毕赤酵母KM71H中诱导表达获得了目的酶蛋白重组β-木糖苷酶(Anxyl); 并将其用于人参皂苷Rb₃及C-Mx的生物转化及催化动力学参数表征. 研究结果表明, Anxyl可将人参皂苷Rb₃和C-Mx分别转化为人参皂苷Rd和C-K. 酶学性质研究表明, 该酶最适反应pH=2.5, 最适反应温度为35 ℃; 在pH=2.0~5.0, 20和30 ℃条件下其具有较好的稳定性, Mg²⁺对酶活具有促进作用. Anxyl对葡萄糖和乙醇有较好的耐受性, 其催化对硝基苯基-β-D-吡喃木糖苷(pNPX)、人参皂苷Rb₃和C-Mx水解的米氏常数(K_m)值分别为3.1, 1.55和1.04 mmol/L, 酶的最大反应速率(V_max)分别为1.9×10², 0.8×10³和8×10³ mmol/min, 酶的转换数(K_cat)值分别为5.55, 0.17和1.85 s^-1, 表明该酶对天然底物Rb₃和C-Mx具有更高的亲和力.

关键词: 黑曲霉, β-木糖苷酶, 人参皂苷转化, 酶动力学

Abstract:

The β-xylosidase gene anxyl which was amplified from Aspergillus niger NG1306 was cloned into pPICZαA to construct the recombinant expression vector(pPICZαA-anxyl). The recombinant protein(Anxyl) was further expressed in Pichia pastoris KM71H for ginsenoside Rb₃, C-Mx biotransformation and catalytic kinetic study. The results showed that anxyl could convert ginsenosides Rb₃ and C-Mx to Rd and C-K, respectively. The results of corresponding enzymatic properties showed that the optimum pH of Anxyl was 2.5, and the optimum reaction temperature was 35 ℃. It has good stability at pH of 2.0—5.0 and 20—30 ℃. Mg ²⁺ could improve the catalytic activity of Anxyl and had good tolerance for glucose and ethanol. The K_mvalues of p-nitrophenyl-β-D-xylopyranoside(pNPX), ginsenoside Rb₃ and C-Mx were 3.1, 1.55 and 1.04 mmol/L, the V_maxvalues of pNPX, Rb₃ and C-Mx were 1.9 10 ², 0.8 10³ and 8 10³ mmol/min, and the K_cat values of pNPX, Rb₃ and C-Mx were 5.55, 0.17 and 1.85 s ^-1, respectively. The results indicated that the recombinant Anxyl had a higher affinity for the natural substrates Rb₃ and C-Mx.

Key words: Aspergillus niger, β-Xylosidase, Ginsenoside biotransformation, Enzyme kinetic

中图分类号:

O629

TrendMD:

朱铃,王雨辰,赵江源,文孟良,李铭刚,韩秀林. 重组β-木糖苷酶转化人参皂苷Rb₃及C-Mx. 高等学校化学学报, 2020, 41(5): 1010.

ZHU Ling,WANG Yuchen,ZHAO Jiangyuan,WEN Mengliang,LI Minggang,HAN Xiulin. Transformation of Ginsenoside Rb₃ and C-Mx by Recombinant β-Xylosidase ^†. Chem. J. Chinese Universities, 2020, 41(5): 1010.

图/表 8

参考文献 39

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Stage	Total activity/U	Total protein mass/mg	Specific activity/ (U·mg^-1)	Yield(%)	Purification fold
Crude extract	1548±0.15	7866±7.89	0.20	100	—
Protein purification	72±0.12	26.84±3.15	50	0.05	253

Stage	Total activity/U	Total protein mass/mg	Specific activity/ (U·mg^-1)	Yield(%)	Purification fold
Crude extract	1548±0.15	7866±7.89	0.20	100	—
Protein purification	72±0.12	26.84±3.15	50	0.05	253

Metal cation/ reagent	Relative enzyme activity(%)
Metal cation/ reagent	10 mmol/L	50 mmol/L	100 mmol/L
NaCl	156±0.02	162±0.08	148±0.08
KCl	123±0.34	119±0.21	125±0.02
MgCl₂	105±0.06	153±0.02	161±0.02
CuCl₂	116±0.07	107±0.06	55±0.15
CaCl₂	92±0.08	90±0.07	98±0.10
CoCl₂	103±10.05	108±0.06	97±0.24
NiCl₂	83±0.21	83±0.29	96±0.08
SDS	118±0.32	98±0.02	33±0.23
EDTA-Na₂	96±0.02	50±0.02	33±0.08
DTT	92±0.05	99±0.09	94±0.01

Metal cation/ reagent	Relative enzyme activity(%)
Metal cation/ reagent	10 mmol/L	50 mmol/L	100 mmol/L
NaCl	156±0.02	162±0.08	148±0.08
KCl	123±0.34	119±0.21	125±0.02
MgCl₂	105±0.06	153±0.02	161±0.02
CuCl₂	116±0.07	107±0.06	55±0.15
CaCl₂	92±0.08	90±0.07	98±0.10
CoCl₂	103±10.05	108±0.06	97±0.24
NiCl₂	83±0.21	83±0.29	96±0.08
SDS	118±0.32	98±0.02	33±0.23
EDTA-Na₂	96±0.02	50±0.02	33±0.08
DTT	92±0.05	99±0.09	94±0.01

Concentration/ (mol·L^-1)	Relative enzyme activity(%)
Concentration/ (mol·L^-1)	D-Xylose	Glucose	Ethanol
0	100±0.08	100±0.02	100±0.05
0.2	59±0.30	99±0.04	Not detected
0.4	57±0.67	96±0.08	Not detected
0.6	51±0.05	95±0.02	Not detected
0.8	7±0.42	95±0.07	99±0.72
1	5±0.05	99±0.04	97±0.13
1.5	Not detected	Not detected	96±0.06
3	Not detected	Not detected	77±0.12
5	Not detected	Not detected	52±0.05
7	Not detected	Not detected	22±0.21

重组β-木糖苷酶转化人参皂苷Rb₃及C-Mx

Transformation of Ginsenoside Rb₃ and C-Mx by Recombinant β-Xylosidase ^†

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