A carbon-carbon hydrolase from human gut probiotics Flavonifractor plautii catalyzes phloretin conversion
A carbon-carbon hydrolase from human gut probiotics Flavonifractor plautii catalyzes phloretin conversion
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DOI：
10.1016/j.fbio.2022.102178
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发表时间：
2022-11
期刊：
影响因子：
5.2
通讯作者：
Tingting Qiu;T. Gong;Shuai Zhang;Q. Chen;C. Hu;Y. Meng
中科院分区：
文献类型：
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作者：
Tingting Qiu;T. Gong;Shuai Zhang;Q. Chen;C. Hu;Y. Meng
虽然一些类黄酮在人类结肠中的分解代谢途径已经被阐明，但对相关肠道微生物和相关酶的了解仍然有限。本研究从普氏黄杆菌(F.plautii)中合成了根素水解酶基因(Phy)，并在大肠杆菌BL21(E.coliBL21)中进行了异源表达。从重组菌细胞提取液中分离纯化了根癌素水解酶(PHY)。ColiBL21，PHY的相对分子质量约为32 kDa。纯化后的PHY能有效地将根癌素切成3-(4-羟基苯基)丙酸(HPPA)和间苯三酚(PG)。测定了PHY的动力学参数：Km为2.8U×10−3±0.4U mm，Vmax为411.2±17.1U/mg。此外，PHY的最适温度和pH分别为50℃和7.5，在50℃下5h后，PHY的活性保持在75%。Ba2+、Cu2+和Fe2+对PHY的活性有抑制作用，而Mg2+对PHY的活性有轻微的促进作用。最后，利用分子对接技术建立了连翘苷与PHY之间的催化模型，并通过多光谱技术进一步研究了PHY的构象变化。本研究有助于了解根癌菌素在结肠中的代谢，为食品工业应用提供有价值的PHY来源。
Although the catabolic pathway of some flavonoids in the human colon has been elucidated, knowledge of related gut microbes and responsible enzymes is still limited. In this study, a phloretin hydrolase gene (phy) fromFlavonifractor plautii(F. plautii) was synthesized and heterologously expressed inEscherichia coliBL21 (E. coliBL 21). Phloretin hydrolase (PHY) was purified from cell extracts of recombinantE. coliBL21, and PHY's molecular weight was about 32 kDa. The purified PHY effectively cleaved phloretin into 3-(4-hydroxyphenyl) propionic acid (HPPA) and phloroglucinol (PG). PHY's kinetic parameters were determined:Kmwas 2.8 × 10−3±0.4 mM andVmaxwas 411.2 ± 7.1 U/mg. Besides, PHY's optimal temperature and pH were 50 °C and 7.5, respectively, and PHY exhibited 75% activity after 5 h at 50 °C. Furthermore, PHY's activity was inhibited in the presence of Ba2+, Cu2+, and Fe2+, while Mg2+slightly elevated PHY's activity. Finally, the catalytic model between phloretin and PHY was built using molecular docking, and PHY's conformational changes were further demonstrated through the multispectral techniques. This study is conducive to understanding phloretin metabolism in the colon and adding a valuable source of PHY for food industry application.