鸡源和猪源乳杆菌胆盐水解酶的表达及酶学性质比较

发布时间：2018-06-07 06:40

  本文选题：胆盐水解酶 + 酶活力　； 参考：《微生物学通报》2017年12期

【摘要】：【目的】随着抗生素生长促进剂(AGPs)在动物饲料中逐步禁止使用,AGPs替代物的研究成为热点。由于胆盐水解酶(BSH)在脂类代谢中的关键作用,成为AGPs替代物研究的一个重要方向。在原核表达和纯化的基础上鉴定鸡源和猪源乳杆菌BSH在酶学性质方面的差异性。【方法】分别对鸡源胆盐水解酶(BSHc)和猪源胆盐水解酶(BSHp)基因进行原核表达和蛋白纯化,通过测定对6种甘氨结合胆盐和牛磺结合胆盐的水解效率获得两种酶的酶学动力学性质,进而测定了温度、pH和金属离子对酶活力的影响。【结果】BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc对甘氨结合胆盐的水解效率较BSHp稍高;BSHc和BSHp的最适酶解温度分别为45°C和42°C;BSHc和BSHp的最适反应pH分别为6.0和5.4;含有Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)的金属盐对BSHc和BSHp的酶活力均具有不同程度的抑制作用,特别是Cu~(2+)和Fe~(3+)抑制作用比较强;含有Na~+、K~+、Mg~(2+)和Ca2+的金属盐对BSHc和BSHp酶活力的抑制作用相对较弱或无抑制作用,但KIO3对BSHc和BSHp酶活力具有强抑制作用,KI和CaCl_2对BSHp酶活力也具有较强的抑制作用。【结论】原核表达和纯化的BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc的最适酶解温度和pH稍高于BSHp,Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)等金属离子对BSHc和BSHp酶活力具有明显抑制作用,试验结果为鉴定BSH抑制物进而研制AGPs替代物奠定了基础。
[Abstract]:[objective] as the antibiotic growth promoter AGPs has gradually banned the use of AGPs substitutes in animal feed, it has become a hot topic. Because of the key role of bile salt hydrolase BSHs in lipid metabolism, BSHs have become an important research direction in the research of AGPs substitutes. On the basis of prokaryotic expression and purification, the differences of enzymatic properties between chicken and porcine Lactobacillus BSH were identified. [methods] the prokaryotic expression and protein purification of chicken bile salt hydrolase BSHc and porcine bile salt hydrolase BSHppc were carried out, respectively. The enzymatic kinetic properties of the two enzymes were obtained by determining the hydrolysis efficiency of 6 kinds of glycine bound bile salts and taurosulfonated bile salts. Furthermore, the effects of temperature pH and metal ions on enzyme activity were determined. [results] the hydrolysis efficiency of BSHc and BSHp on glycosamino-bound bile salt was higher than that of tauro-bound bile salt BSHc on glycosamino-bound bile salt, which was slightly higher than that of BSHp and BSHp. The optimum pH for enzymatic hydrolysis at 45 掳C and 42 掳C for BSHc and BSHp were 6.0 and 5.4, respectively. Metal salts containing Cu~(2 and Zn~(2) inhibited the enzyme activities of BSHc and BSHp in different degrees. In particular, Cu~(2) and Fe~(3) had strong inhibitory effects on the enzyme activities of BSHc and BSHp, and the metal salts containing Na ~ + K ~ (2) and Ca2 had relatively weak or no inhibitory effects on the activities of BSHc and BSHp. But KIO3 can inhibit the activity of BSHc and BSHp. Ki and CaCl_2 have strong inhibition on BSHp activity. [conclusion] the hydrolytic efficiency of prokaryotic expression and purified BSHc and BSHp is higher than that of taurocholate. [conclusion] the hydrolysis efficiency of prokaryotic expression and purified BSHc and BSHp is higher than that of taurocholate. The optimum enzymatic hydrolysis temperature and pH of BSHc were slightly higher than those of BSHpCuO2) and Zn~(2), and the activities of BSHc and BSHp were inhibited obviously. The results laid a foundation for the identification of BSH inhibitors and the development of AGPs substitutes.
【作者单位】： 北京市农林科学院畜牧兽医研究所畜禽疫病防控技术北京市重点实验室;
【基金】：国家自然科学基金项目(No.31572527) 北京市农林科学院科技创新能力建设专项项目(No.KJCX20150703,KJCX20161503)~~
【分类号】：S816.3

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