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Project","award":["2023AH010045"],"award-info":[{"award-number":["2023AH010045"]}]},{"name":"Anhui Province University Innovation Team Project","award":["GXXT-2023-107"],"award-info":[{"award-number":["GXXT-2023-107"]}]},{"name":"Anhui Province University Innovation Team Project","award":["2024340603000274"],"award-info":[{"award-number":["2024340603000274"]}]},{"name":"Anhui Province University Innovation Team Project","award":["2024019"],"award-info":[{"award-number":["2024019"]}]},{"name":"Anhui Province University Innovation Team Project","award":["2208085MC65"],"award-info":[{"award-number":["2208085MC65"]}]},{"name":"Anhui Province University Innovation Team Project","award":["gxgnfx2021118"],"award-info":[{"award-number":["gxgnfx2021118"]}]},{"name":"Anhui Province University Innovation Team Project","award":["2023AH010045"],"award-info":[{"award-number":["2023AH010045"]}]}],"content-domain":{"domain":["www.mdpi.com"],"crossmark-restriction":true},"short-container-title":["Microorganisms"],"abstract":"<jats:p>\u03b2-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 \u03b2-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 \u00b0C, and it rapidly lost activity at pH &lt; 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 \u03b2-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 \u00b0C, 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 (AlphaFold v2.0, DeepMind Technologise Limited, London, UK), and p-Nitrophenyl-\u03b2-D-Glucuronide (pNPG) and conjugated bilirubin were then docked to the CpGUS protein model using SWISSDOCK. The best docked conformations of the CpGUS\u2013pNPG and CpGUS\u2013conjugated 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 \u03c0-\u03c0 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 \u03c0-\u03c0 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 (\u2206Gbind) values were calculated to be as high as \u221265.05 \u00b1 12.66 and \u221286.70 \u00b1 17.18 kJ\/mol, respectively. These results suggest that CpGUS possesses high binding and catalytic hydrolysis properties for both pNPG and conjugated bilirubin.<\/jats:p>","DOI":"10.3390\/microorganisms13051043","type":"journal-article","created":{"date-parts":[[2025,5,1]],"date-time":"2025-05-01T05:10:32Z","timestamp":1746076232000},"page":"1043","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Heterologous Expression and Enzymatic Properties of \u03b2-Glucuronidase from Clostridium perfringens and Its Application in Bilirubin Transformation"],"prefix":"10.3390","volume":"13","author":[{"given":"Qianlin","family":"Wu","sequence":"first","affiliation":[{"name":"Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei 235000, China"},{"name":"School of Life Sciences, Huaibei Normal University, Huaibei 235000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qing","family":"Guo","sequence":"additional","affiliation":[{"name":"Anhui Chem-Bright Bioengineering Co., Ltd., Huaibei 235000, China"},{"name":"Anhui Province Engineering Technology Research Center for Livestock by-Product Medical Intermediate Extraction, Huaibei 235000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fo","family":"Yang","sequence":"additional","affiliation":[{"name":"Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei 235000, China"},{"name":"School of Life Sciences, Huaibei Normal University, Huaibei 235000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengru","family":"Li","sequence":"additional","affiliation":[{"name":"Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei 235000, China"},{"name":"School of Life Sciences, Huaibei Normal University, Huaibei 235000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yumeng","family":"Zhu","sequence":"additional","affiliation":[{"name":"Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei 235000, China"},{"name":"School of Life Sciences, Huaibei Normal University, Huaibei 235000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Binpeng","family":"Xu","sequence":"additional","affiliation":[{"name":"Anhui Chem-Bright Bioengineering Co., Ltd., Huaibei 235000, China"},{"name":"Anhui Province Engineering Technology Research Center for Livestock by-Product Medical Intermediate Extraction, Huaibei 235000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lu","family":"Zhao","sequence":"additional","affiliation":[{"name":"Anhui Chem-Bright Bioengineering Co., Ltd., Huaibei 235000, China"},{"name":"Anhui Province Engineering Technology Research Center for Livestock by-Product Medical Intermediate Extraction, Huaibei 235000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shanshan","family":"Zhang","sequence":"additional","affiliation":[{"name":"Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei 235000, China"},{"name":"School of Life Sciences, Huaibei Normal University, Huaibei 235000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Youyu","family":"Xie","sequence":"additional","affiliation":[{"name":"Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei 235000, China"},{"name":"School of Life Sciences, Huaibei Normal University, Huaibei 235000, 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235000, China"},{"name":"School of Life Sciences, Huaibei Normal University, Huaibei 235000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,4,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"637","DOI":"10.1016\/S0959-440X(97)80072-3","article-title":"Structural and sequence-based classification of glycoside hydrolases","volume":"7","author":"Henrissat","year":"1997","journal-title":"Curr. 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