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Frontiers of Chemical Science and Engineering

Front. Chem. Sci. Eng.    2019, Vol. 13 Issue (3) : 554-562
Construction of a CaHPO4-PGUS1 hybrid nanoflower through protein-inorganic self-assembly, and its application in glycyrrhetinic acid 3-O-mono-β-D-glucuronide preparation
Tian Jiang, Yuhui Hou, Tengjiang Zhang, Xudong Feng(), Chun Li()
Institute for Synthetic Biosystem/Department of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, China
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Glycyrrhetinic acid 3-O-mono-b-D-glucuronide (GAMG), an important pharmaceutical intermediate and functional sweetener, has broad applications in the food and medical industries. A green and cost-effective method for its preparation is highly desired. Using site-directed mutagenesis, we previously obtained a variant of β-glucuronidase from Aspergillus oryzae Li-3 (PGUS1), which can specifically transform glycyrrhizin (GL) into GAMG. In this study, a facile method was established to prepare a CaHPO4-PGUS1 hybrid nanoflower for enzyme immobilization, based on protein-inorganic hybrid self-assembly. Under optimal conditions, 1.2 mg of a CaHPO4-PGUS1 hybrid nanoflower precipitate with 71.2% immobilization efficiency, 35.60 mg·g−1 loading capacity, and 118% relative activity was obtained. Confocal laser scanning microscope and scanning electron microscope results showed that the enzyme was encapsulated in the CaHPO4-PGUS1 hybrid nanoflower. Moreover, the thermostability of the CaHPO4-PGUS1 hybrid nanoflower at 55°C was improved, and its half-life increased by 1.3 folds. Additionally, the CaHPO4-PGUS1 hybrid nanoflower was used for the preparation of GAMG through GL hydrolysis, with the conversion rate of 92% in 8 h, and after eight consecutive runs, it had 60% of its original activity.

Keywords β-glucuronidase      enzyme-inorganic hybrid nanoflower      biotransformation      glycyrrhizin      glycyrrtinic acid 3-O-mono-β-D-glucuronide     
Corresponding Authors: Xudong Feng,Chun Li   
Just Accepted Date: 06 June 2019   Online First Date: 24 July 2019    Issue Date: 22 August 2019
 Cite this article:   
Tian Jiang,Yuhui Hou,Tengjiang Zhang, et al. Construction of a CaHPO4-PGUS1 hybrid nanoflower through protein-inorganic self-assembly, and its application in glycyrrhetinic acid 3-O-mono-β-D-glucuronide preparation[J]. Front. Chem. Sci. Eng., 2019, 13(3): 554-562.
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Tian Jiang
Yuhui Hou
Tengjiang Zhang
Xudong Feng
Chun Li
Fig.1  Effect of divalent metal ions on the activity of PGUS1.
Fig.2  Optimizing the conditions for CaHPO4-PGUS1 hybrid nanoflower preparation by controlling the concentration of (a) Ca2+, (b) phosphate, and (c) PGUS1, and (d) pH.
Fig.3  Effect of incubation time and temperature on CaHPO4-PGUS1 hybrid nanoflower formation: (a) mass, (b) loading capacity, (c) relative activity.
Ca2+/(mmol?L?1) Phosphate/(mmol?L?1) PGUS1/(mg·mL–1) pH Time/h Temperature/°C
1.6 8 0.4 6.5 12 4
Tab.1  Optimal conditions for CaHPO4-PGUS1 hybrid nanoflower preparation
Amount of nanoflowers
Immobilization efficiency
Loading capacity
Relative activity
1.2 71.2 35.60 118
Tab.2  Critical parameters of CaHPO4-PGUS1 hybrid nanoflower prepared under optimal conditions
Fig.4  Characterization of CaHPO4-PGUS1 hybrid nanoflower: (a, b) SEM images, (c) CLSM image.
Zeta potential/mV Conductivity/(mS?cm–1) Particle size/µm
CaHPO4 ?19.1±3.2 4.9±0.4 Nda
Nanoflower ?10.8±0.3 1.0±0.04 3.6±0.4
Tab.3  The zeta potential, conductivity and particle size of CaHPO4-PGUS1 hybrid nanoflower
Enzyme Km/(mmol?L?1) kcat/s–1 kcat/Km (L?mmol–1?s–1)
Free PGUS1 2.51±0.05 5.67±0.05 2.26
Nanoflower 1.47±0.02 1.65±0.02 1.22
Tab.4  Kinetic parameters of free PGUS1 and CaHPO4-PGUS1 hybrid nanoflower
Fig.5  (a) Thermostability and (b) thermal deactivation of CaHPO4-PGUS1 hybrid nanoflower and free PGUS1 at 55°C in 10 h. Values are the average of 3 independent experiments and error bars represent average ±1 S.D.
Fig.6  The application of CaHPO4-PGUS1 hybrid nanoflower in GL transformation: (a) Course of the biotransformation of GL mediated by free PGUS1 and CaHPO4-PGUS1 hybrid nanoflower (CaHPO4-PGUS1 hybrid nanoflower precipitate (170 mg) and equal amount of free enzyme was used); (b) Reusability of CaHPO4-PGUS1 hybrid nanoflower.
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