Yadav Ruby, Singh Puneet K, Puniya Anil K, Shukla Pratyoosh
Enzyme Technology and Protein Bioinformatics Laboratory, Department of Microbiology, Maharshi Dayanand University Rohtak, India.
Division of Dairy Microbiology, Indian Council of Agricultural Research (ICAR) - National Dairy Research Institute (NDRI)Karnal, India; College of Dairy Science and Technology, Guru Angad Dev Veterinary and Animal Sciences UniversityLudhiana, India.
Front Microbiol. 2017 Jan 5;7:2116. doi: 10.3389/fmicb.2016.02116. eCollection 2016.
Prebiotics are the non-digestible carbohydrate, which passes through the small intestine into unmetabolized form, reaches the large intestine and undergoes fermentation by the colonic bacteria thus; prebiotics stimulate the growth of probiotic bacteria. Further, bile salt hydrolase (BSH) is an enzyme that catalyses the deconjugation of bile salt, so it has enormous potential toward utilizing such capability of RYPR1 toward detoxifying through BSH enzyme activity. In the present study, six isolates of were evaluated for the co-aggregation assay and the isolate RYPR1 was further selected for studies of prebiotic utilization, catalytic interactions and molecular docking. The prebiotic utilization ability was assessed by using commercially available prebiotics lactulose, inulin, xylitol, raffinose, and oligofructose P95. The results obtained revealed that RYPR1 is able to utilize these probiotics, maximum with lactulose by showing an increase in viable cell count (7.33 ± 0.02 to 8.18 ± 0.08). In addition, the molecular docking of BSH from RYPR1 was performed which revealed the binding energy -4.42 and 7.03 KJ/mol. This proves a considerably good interactions among BSH and its substrates like Taurocholic acid (-4.42 KJ/mol) and Glycocholic acid (-7.03 KJ/mol). These results from this study establishes that RYPR1 possesses good probiotic effects so it could be used for such applications. Further, molecular dynamics simulations were used to analyze the dynamic stability of the of modeled protein to stabilize it for further protein ligand docking and it was observed that residues Asn12, Ile8, and Leu6 were interacting among BSH and its substrates, i.e., Taurocholic acid and Lys88 and Asp126 were interacting with Glycocholic acid. These residues were interacting when the docking was carried out with stabilized BSH protein structure, thus, these residues may have a vital role in stabilizing the binding of the ligands with the protein.
益生元是不可消化的碳水化合物，它以未代谢的形式穿过小肠，到达大肠并被结肠细菌发酵；因此，益生元能刺激益生菌的生长。此外，胆盐水解酶（BSH）是一种催化胆汁盐去结合的酶，所以利用RYPR1通过BSH酶活性进行解毒的这种能力具有巨大潜力。在本研究中，对六种分离株进行了共聚集分析，并进一步选择分离株RYPR1进行益生元利用、催化相互作用和分子对接研究。通过使用市售的益生元乳果糖、菊粉、木糖醇、棉子糖和低聚果糖P95来评估益生元利用能力。所得结果表明，RYPR1能够利用这些益生菌，对乳果糖的利用效果最佳，活菌数增加（从7.33±0.02增加到8.18±0.08）。此外，对RYPR1的BSH进行了分子对接，结果显示结合能为-4.42和7.03千焦/摩尔。这证明了BSH与其底物如牛磺胆酸（-4.42千焦/摩尔）和甘氨胆酸（-7.03千焦/摩尔）之间有相当良好的相互作用。本研究的这些结果表明，RYPR1具有良好的益生菌效果，因此可用于此类应用。此外，分子动力学模拟用于分析建模蛋白质的动态稳定性，以使其稳定以便进一步进行蛋白质配体对接，观察到Asn12、Ile8和Leu6残基在BSH与其底物（即牛磺胆酸）之间相互作用，而Lys88和Asp126与甘氨胆酸相互作用。当用稳定的BSH蛋白质结构进行对接时，这些残基会相互作用，因此，这些残基可能在稳定配体与蛋白质的结合中起重要作用。
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