Ⅱ型丙酮酸激酶的表达与功能研究
发布时间：2018-10-25 08:53
【摘要】：三磷酸腺苷(Adenosine triphosphate,ATP)在医疗领域应用广泛,对肝炎、脑溢血后遗症、心肌疾患等多种疾病均具有良好疗效。但由于三磷酸腺苷酶法合成中酶的价格昂贵,未在工业上获得广泛应用。在生物体内外Mg2+的存在下,丙酮酸激酶(Pyruvate kinase,PK)能够催化磷酸烯醇丙酮酸(PEP)的磷酸基团转移到二磷酸腺苷(ADP)上从而生成丙酮酸(PYR)和ATP。此步反应为ATP的酶法合成提供了新的思路;而且基于此步反应的ATP再生系统,采用再生ATP的廉价底物来替代ATP,可以有效提高有用物质生产过程的经济性。本研究利用DNAMAN、MEGA 7软件对Pyk A氨基酸序列进行分析发现,Pyk A中涉及构成ATP及PEP结合位点的氨基酸残基是高度保守的。然后进一步采用基因工程技术成功构建Ⅱ型丙酮酸激酶(PykA)的表达载体pET28a(+)-pykA,并实现了其在大肠杆菌BL21中的高效表达:1L菌液可以得到34mg的蛋白;利用镍柱对PykA进行纯化,并对纯化后的蛋白进行定量,浓度可达到2.3mg/mL。通过高效液相色谱技术(HPLC)对Pyk A催化功能进行检测,结果发现在30℃,pH为8.0的环境中,ADP和PEP的浓度比为1:1时,无论反应进行0.5h还是1h,0.05mg/mL的PykA都无法将ADP完全转化为ATP,ATP的积累对PykA催化ADP磷酸化反应具有抑制作用;当ADP和PEP的浓度比为1:2时,0.05mg/m L的Pyk A只需0.5h就可以将ADP完全转化为ATP。本研究的结果可以达到将丙酮酸激酶更好地应用于ATP再生系统,为ATP的酶法合成提供新的思路的目的。
[Abstract]:Adenosine triphosphate (Adenosine triphosphate,ATP) is widely used in medical field and has good curative effect on hepatitis, sequelae of cerebral hemorrhage and myocardial diseases. However, because of the high price of adenosine triphosphate enzyme synthesis, it has not been widely used in industry. In the presence of Mg2 in vivo and in vitro, pyruvate kinase (Pyruvate kinase,PK) can catalyze the transfer of phosphate groups from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP) to produce pyruvate (PYR) and ATP.. This step provides a new idea for the enzymatic synthesis of ATP, and the ATP regeneration system based on this step can effectively improve the economy of the production process of useful substances by replacing ATP, with the cheap substrate of regenerated ATP. In this study, DNAMAN,MEGA 7 software was used to analyze the amino acid sequence of Pyk A. It was found that the amino acid residues involved in ATP and PEP binding sites in, Pyk A were highly conserved. Then the expression vector pET28a ()-pykA, of type 鈪，
本文编号：2293219
[Abstract]:Adenosine triphosphate (Adenosine triphosphate,ATP) is widely used in medical field and has good curative effect on hepatitis, sequelae of cerebral hemorrhage and myocardial diseases. However, because of the high price of adenosine triphosphate enzyme synthesis, it has not been widely used in industry. In the presence of Mg2 in vivo and in vitro, pyruvate kinase (Pyruvate kinase,PK) can catalyze the transfer of phosphate groups from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP) to produce pyruvate (PYR) and ATP.. This step provides a new idea for the enzymatic synthesis of ATP, and the ATP regeneration system based on this step can effectively improve the economy of the production process of useful substances by replacing ATP, with the cheap substrate of regenerated ATP. In this study, DNAMAN,MEGA 7 software was used to analyze the amino acid sequence of Pyk A. It was found that the amino acid residues involved in ATP and PEP binding sites in, Pyk A were highly conserved. Then the expression vector pET28a ()-pykA, of type 鈪，
本文编号：2293219
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