【摘要】：三磷酸腺苷(Adenosine triphosphate,ATP)在醫(yī)療領(lǐng)域應(yīng)用廣泛,對(duì)肝炎、腦溢血后遺癥、心肌疾患等多種疾病均具有良好療效。但由于三磷酸腺苷酶法合成中酶的價(jià)格昂貴,未在工業(yè)上獲得廣泛應(yīng)用。在生物體內(nèi)外Mg2+的存在下,丙酮酸激酶(Pyruvate kinase,PK)能夠催化磷酸烯醇丙酮酸(PEP)的磷酸基團(tuán)轉(zhuǎn)移到二磷酸腺苷(ADP)上從而生成丙酮酸(PYR)和ATP。此步反應(yīng)為ATP的酶法合成提供了新的思路;而且基于此步反應(yīng)的ATP再生系統(tǒng),采用再生ATP的廉價(jià)底物來(lái)替代ATP,可以有效提高有用物質(zhì)生產(chǎn)過(guò)程的經(jīng)濟(jì)性。本研究利用DNAMAN、MEGA 7軟件對(duì)Pyk A氨基酸序列進(jìn)行分析發(fā)現(xiàn),Pyk A中涉及構(gòu)成ATP及PEP結(jié)合位點(diǎn)的氨基酸殘基是高度保守的。然后進(jìn)一步采用基因工程技術(shù)成功構(gòu)建Ⅱ型丙酮酸激酶(PykA)的表達(dá)載體pET28a(+)-pykA,并實(shí)現(xiàn)了其在大腸桿菌BL21中的高效表達(dá):1L菌液可以得到34mg的蛋白;利用鎳柱對(duì)PykA進(jìn)行純化,并對(duì)純化后的蛋白進(jìn)行定量,濃度可達(dá)到2.3mg/mL。通過(guò)高效液相色譜技術(shù)(HPLC)對(duì)Pyk A催化功能進(jìn)行檢測(cè),結(jié)果發(fā)現(xiàn)在30℃,pH為8.0的環(huán)境中,ADP和PEP的濃度比為1:1時(shí),無(wú)論反應(yīng)進(jìn)行0.5h還是1h,0.05mg/mL的PykA都無(wú)法將ADP完全轉(zhuǎn)化為ATP,ATP的積累對(duì)PykA催化ADP磷酸化反應(yīng)具有抑制作用;當(dāng)ADP和PEP的濃度比為1:2時(shí),0.05mg/m L的Pyk A只需0.5h就可以將ADP完全轉(zhuǎn)化為ATP。本研究的結(jié)果可以達(dá)到將丙酮酸激酶更好地應(yīng)用于ATP再生系統(tǒng),為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，

本文編號(hào)：2293220