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

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.

Graphical abstract

Keywords

β-glucuronidase / enzyme-inorganic hybrid nanoflower / biotransformation / glycyrrhizin / glycyrrtinic acid 3-O-mono-β-D-glucuronide

References

[1] Asl M N, Hosseinzadeh H. Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds. Phytotherapy Research, 2010, 22(6): 709–724

[2] Cinatl J, Morgenstern B, Bauer G, Chandra P, Rabenau H, Doerr H W. Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet, 2003, 361(9374): 2045–2046

[3] Baltina L A. Chemical modification of glycyrrhizic acid as a route to new bioactive compounds for medicine. Current Medicinal Chemistry, 2003, 10(2): 155–171

[4] Zhao Y, Lv B, Feng X D, Li C. Perspective on biotransformation and de novo biosynthesis of licorice constituents. Journal of Agricultural and Food Chemistry, 2017, 65(51): 11147–11156

[5] Amin H A S, El-Menoufy H A, El-Mehalawy A A, Mostafa E S. Biosynthesis of glycyrrhetinic acid 3-O-mono-β-D-glucuronide by free and immobilized Aspergillus terreus β-D-glucuronidase. Journal of Molecular Catalysis B: Enzymatic, 2011, 69: 54–59

[6] Lu D Q, Li H, Dai Y, Ouyang P K. Biocatalytic properties of a novel crude glycyrrhizin hydrolase from the liver of the domestic duck. Journal of Molecular Catalysis B: Enzymatic, 2006, 43: 148–152

[7] Kim D H, Lee S W, Han M J. Biotransformation of glycyrrhizin to 18 β-glycyrrhetinic acid-3-O-β-D-glucuronide by Streptococcus LJ-22, a human intestinal bacterium. Biological & Pharmaceutical Bulletin, 1999, 22: 320–322

[8] Wang X, Liu Y Q, Wang C X, Feng X D, Li C. Properties and structures of β-glucuronidases with different transformation types of glycyrrhizin. RSC Advances, 2015, 5(84): 68345–68350

[9] Lv B, Sun H, Huang S, Feng X, Jiang T, Li C. Structure-guided engineering of the substrate specificity of a fungal β-glucuronidase toward triterpenoid saponins. Journal of Biological Chemistry, 2018, 293(2): 433–443

[10] Han B, Hou Y, Jiang T, Lv B, Zhao L, Feng X, Li C. Computation-aided rational deletion of C-terminal region improved the stability, activity and expression level of GH₂ β-glucuronidase. Journal of Agricultural and Food Chemistry, 2018, 66(43): 11380–11389

[11] Guzik U, Hupert-Kocurek K, Wojcieszyåska D. Immobilization as a strategy for improving enzyme properties-application to oxidoreductases. Molecules (Basel, Switzerland), 2014, 19(7): 8995–9018

[12] Hou C, Wang Y, Zhu H, Wei H. Construction of enzyme immobilization system through metal-polyphenol assisted Fe₃O₄/chitosan hybrid microcapsules. Chemical Engineering Journal, 2016, 283: 397–403

[13] Song X, Jiang Z, Li L, Wu H. Immobilization of β-glucuronidase in lysozyme-induced biosilica particles to improve its stability. Frontiers of Chemical Science and Engineering, 2014, 8(3): 353–361

[14] Jesionowski T, Zdarta J, Krajewska B. Enzyme immobilization by adsorption: A review. Adsorption, 2014, 20(5-6): 801–821

[15] Yang X Y, Guo Y S, Zhang S S. Ultrasensitive enhanced chemiluminescence enzyme immunoassay for the determination of alpha-fetoprotein amplified by double-codified gold nanoparticles labels. Biosensors & Bioelectronics, 2009, 24(8): 2707–2711

[16] Ge J, Lei J, Zare R N. Protein-inorganic hybrid nanoflowers. Nature Nanotechnology, 2012, 7(7): 428–432

[17] Wu Z, Li X, Li F, Yue H, He C, Wang Z. Enantioselective transesterification of (R,S)-2-pentanol catalyzed by a new flower-like nanobioreactor. RSC Advances, 2014, 4(64): 33998–34002

[18] Wang L B, Wang Y C, He R, Zhuang A, Wang X, Zeng J, Hou J G. A new nanobiocatalytic system based on allosteric effect with dramatically enhanced enzymatic performance. Journal of the American Chemical Society, 2013, 135(4): 1272–1275

[19] Lin Z, Xiao Y, Yin Y, Hu W, Liu W, Yang H. Facile synthesis of enzyme-inorganic hybrid nanoflowers and its application as a colorimetric platform for visual detection of hydrogen peroxide and phenol. ACS Applied Materials & Interfaces, 2014, 6(13): 10775–10782

[20] Bradford M M. Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Analytical Biochemistry, 1976, 72(1-2): 248–254

[21] Henley J P, Sadana A. Deactivation theory. Biotechnology and Bioengineering, 1986, 28(8): 1277–1285

[22] Duan Y, Liu X, Han L, Asahina S, Xu D, Cao Y, Yao Y, Che S. Optically active chiral CuO “nanoflowers”. Journal of the American Chemical Society, 2014, 136(20): 7193–7196

[23] Zeng J, Xia Y. Hybrid nanomaterials. Not just a pretty flower. Nature Nanotechnology, 2012, 7(7): 415–416

[24] Sun J, Ge J, Liu W, Lan M, Zhang H, Wang P, Wang Y, Niu Z. Multi-enzyme co-embedded organic-inorganic hybrid nanoflowers: Synthesis and application as a colorimetric sensor. Nanoscale, 2014, 6(1): 255–262

[25] Lin Z, Xiao Y, Wang L, Yin Y, Zheng J, Yang H, Chen G. Facile synthesis of enzyme-inorganic hybrid nanoflowers and their application as an immobilized trypsin reactor for highly efficient protein digestion. RSC Advances, 2014, 4(27): 13888–13891

[26] Chen S, Shi X, Osaka A, Gao H, Hanagata N. Facile synthesis, microstructure and BMP-2 delivery of novel silica hollow flowers for enhanced osteoblast differentiation. Chemical Engineering Journal, 2014, 246: 1–9

[27] Wang X, Shi J, Li Z, Zhang S, Wu H, Jiang Z, Yang C, Tian C. Facile one-pot preparation of chitosan/calcium pyrophosphate hybrid microflowers. ACS Applied Materials & Interfaces, 2014, 6: 14522–14532

[28] Feng X, Patterson D, Balaban M, Emanuelssom E. Characterization of tributyrin hydrolysis by immobilized lipase on woolen cloth using conventional batch and novel spinning cloth disc reactors. Chemical Engineering Research & Design, 2013, 91(9): 1684–1692

[29] Chen L, Wei B, Zhang X, Li C. Bifunctional graphene/γ-Fe₂O₃ hybrid aerogels with double nanocrystalline networks for enzyme immobilization. Small, 2013, 9(13): 2331–2340

[30] Kaleem I, Rasool A, Lv B, Riaz N, Hassan J U, Manzoor R, Li C. Immobilization of purified β-glucuronidase on ZnO nanoparticles for efficient biotransformation of glycyrrhizin in ionic liquid/buffer biphasic system. Chemical Engineering Science, 2017, 162: 332–340

Acknowledgments

This research was funded by the National Natural Science Foundation of China (Grant Nos. 21425624, 21878021, and 21506011).

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2019 Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature