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Article

Heterologous Expression and Enzymatic Properties of β-Glucuronidase from Clostridium perfringens and Its Application in Bilirubin Transformation

1
Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei 235000, China
2
School of Life Sciences, Huaibei Normal University, Huaibei 235000, China
3
Anhui Chem-Bright Bioengineering Co., Ltd., Huaibei 235000, China
4
Anhui Province Engineering Technology Research Center for Livestock By-Product Medical Intermediate Extraction, Huaibei 235000, China
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Microorganisms 2025, 13(5), 1043; https://doi.org/10.3390/microorganisms13051043
Submission received: 17 March 2025 / Revised: 24 April 2025 / Accepted: 28 April 2025 / Published: 30 April 2025
(This article belongs to the Section Molecular Microbiology and Immunology)

Abstract

β-glucuronidase is an important hydrolase, which plays an important role in drug metabolism, clinical diagnostics, and biotransformation. This study focuses on the heterologous expression, isolation, purification, and its enzymatic properties of β-glucuronidase CpGUS from Clostridium perfringens, as well as its application in the whole-cell transformation of unconjugated bilirubin from pig bile. A recombinant E. coli BL21(DE3)/pET-28a-CpGUS was constructed for the heterologous expression of CpGUS, with the majority of the expressed enzyme being soluble. Enzymatic analysis showed that CpGUS displayed optimal activity at pH 5.0 and 45 °C, and it rapidly lost activity at pH < 4.5. Metal ions, such as Mg2+ and Fe2+, enhanced CpGUS catalysis, while Zn2+, K+, Fe3+, Mn2+, Cu2+, and Na+ inhibited it. Notably, Cu²⁺ and Fe³⁺ can significantly inhibit β-glucuronidase, resulting in the complete loss of its activity. The results of the whole-cell transformation experiment show that when E.coli BL21(DE3)/ pET-28a-CpGUS at an OD600 of 10 was incubated at pH 5.0, a temperature of 45 °C, and a rotation speed of 200 rpm for 12 h, the hydrolysis rate of the conjugated bilirubin in pig bile reached 81.1%, the yield of unconjugated bilirubin was 76.8%, and the purity of unconjugated bilirubin was 98.2%. The three-dimensional structure of CpGUS was predicted using AlphaFold2, and p-Nitrophenyl-β-D-Glucuronide (pNPG) and conjugated bilirubin were then docked to the CpGUS protein model using SWISSDOCK. The best docked conformations of the CpGUS–pNPG and CpGUS–conjugated bilirubin complex systems were simulated by independent 500 ns molecular dynamics (MD) runs with the RSFF2C force field, and the binding dynamic and catalytic mechanism of each system were obtained. The results indicated that π-π stacking, hydrogen bonding, and hydrophobic interactions between the key residue Tyr472 and the benzene ring of pNPG molecules are crucial for its catalytic process. Similarly, for the binding and catalysis of conjugated bilirubin by CpGUS, the π-π stacking and hydrogen bonding and hydrophobic interactions between the sidechains of residues Phe368 and Tyr472 and the benzene ring of conjugated bilirubin play a synergistic role during its catalytic process. Their total binding free energy (∆Gbind) values were calculated to be as high as −65.05 ± 12.66 and −86.70 ± 17.18 kJ/mol, respectively. These results suggest that CpGUS possesses high binding and catalytic hydrolysis properties for both pNPG and conjugated bilirubin.
Keywords: β-glucuronidase CpGUS; bilirubin; molecular dynamics simulation; catalytic mechanism β-glucuronidase CpGUS; bilirubin; molecular dynamics simulation; catalytic mechanism

Share and Cite

MDPI and ACS Style

Wu, Q.; Guo, Q.; Yang, F.; Li, M.; Zhu, Y.; Xu, B.; Zhao, L.; Zhang, S.; Xie, Y.; Li, F.; et al. Heterologous Expression and Enzymatic Properties of β-Glucuronidase from Clostridium perfringens and Its Application in Bilirubin Transformation. Microorganisms 2025, 13, 1043. https://doi.org/10.3390/microorganisms13051043

AMA Style

Wu Q, Guo Q, Yang F, Li M, Zhu Y, Xu B, Zhao L, Zhang S, Xie Y, Li F, et al. Heterologous Expression and Enzymatic Properties of β-Glucuronidase from Clostridium perfringens and Its Application in Bilirubin Transformation. Microorganisms. 2025; 13(5):1043. https://doi.org/10.3390/microorganisms13051043

Chicago/Turabian Style

Wu, Qianlin, Qing Guo, Fo Yang, Mengru Li, Yumeng Zhu, Binpeng Xu, Lu Zhao, Shanshan Zhang, Youyu Xie, Feng Li, and et al. 2025. "Heterologous Expression and Enzymatic Properties of β-Glucuronidase from Clostridium perfringens and Its Application in Bilirubin Transformation" Microorganisms 13, no. 5: 1043. https://doi.org/10.3390/microorganisms13051043

APA Style

Wu, Q., Guo, Q., Yang, F., Li, M., Zhu, Y., Xu, B., Zhao, L., Zhang, S., Xie, Y., Li, F., Wu, X., & Xu, D. (2025). Heterologous Expression and Enzymatic Properties of β-Glucuronidase from Clostridium perfringens and Its Application in Bilirubin Transformation. Microorganisms, 13(5), 1043. https://doi.org/10.3390/microorganisms13051043

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