鸡源和猪源乳杆菌胆盐水解酶的表达及酶学性质比较

Expression and characterization of bile salt hydrolase from chicken and porcine Lactobacillus

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Cu2+、Fe3+、Mn2+和Zn2+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Cu2+和Fe3+抑制作用比较强；含有Na+、K+、Mg2+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CaCl2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，Cu2+、Fe3+、Mn2+和Zn2+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] With the gradual prohibition of the use of antibiotic growth promoters (AGPs) in animal feed, studies on AGPs alternatives are becoming hot topics. As the bile salt hydrolase plays a key role in lipid metabolism, it has become an important direction in AGPs alternatives. This study is aimed to determine the difference in enzymatic properties of bile salt hydrolase from chicken and porcine Lactobacillus based on the prokaryotic expression and purification. [Methods] The encoding genes of chicken bile salt hydrolase (BSHc) and porcine bile salt hydrolase (BSHp) were expressed in E. coli and purified by His-tag affinity column chromatography. The purified products were used to identify the BSH kinetic properties by hydrolyzing the six glycoconjugated and tauroconjugated bile salts. Effects of temperature, pH and metal ion compounds on the BSH activity were also determined respectively. [Results] BSHc and BSHp displayed higher catalytic efficiencies on glycoconjugated bile salts than that of tauroconjugated bile salts, while BSHc had a slightly higher hydrolysis activity on glycoconjugated bile salts than BSHp. The higher enzymatic activity of BSHc and BSHp were observed at the temperature of 45 °C and 42 °C, respectively. The optimal pH for BSHc and BSHp were 6.0 and 5.4, respectively. The metal ion compounds containing Cu2+, Fe3+, Mn2+, and Zn2+ displayed different degrees of inhibition on BSHc and BSHp activity, especially higher inhibition observed in Cu2+ and Fe3+ compounds. The inhibition of the compounds containing Na+, K+, Mg2+, and Ca2+ on BSHc and BSHp activity was relatively weak or no inhibition, but KIO3 had a strong inhibitory effect on BSHc and BSHp activity, KI and CaCl2 also had strong inhibitory effects on BSHp activity. [Conclusion] Based on prokaryotic expression and protein purification, BSHc and BSHp displayed higher catalytic efficiencies on glycoconjugated bile salts than that of tauroconjugated bile salts. The optimal temperature and pH for BSHc was higher than that for BSHp. The metal ion compounds containing Cu2+, Fe3+, Mn2+, and Zn2+ displayed significant inhibition on BSHc and BSHp activity. The results will be helpful for identification of BSH inhibitors and development of AGPs alternatives.