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

doi:10.1007/s11705-019-1834-z

Front. Chem. Sci. Eng.

2019, Vol. 13

Issue (3) : 554-562 https://doi.org/10.1007/s11705-019-1834-z

RESEARCH ARTICLE

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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Abstract

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 Author(s): 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.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-019-1834-z
https://academic.hep.com.cn/fcse/EN/Y2019/V13/I3/554

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.

Tab.1 Optimal conditions for CaHPO₄-PGUS1 hybrid nanoflower preparation

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.

Tab.3 The zeta potential, conductivity and particle size of CaHPO₄-PGUS1 hybrid nanoflower

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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