来源于泗阳鞘氨醇杆菌的聚磷酸激酶的克隆表达及在ATP再生系统中的应用

Expression of polyphosphate kinase from Sphingobacterium siyangensis and its application in ATP regeneration system

摘要:

聚磷酸激酶(polyphosphate kinase,PPK)在体外催化合成ATP的反应中有着重要作用。为寻找能利用短链聚磷酸盐(polyphosphate,polyP)为底物高效合成ATP的聚磷酸激酶，本文以来源于泗阳鞘氨醇杆菌(Sphingobacterium siyangensis)的聚磷酸激酶(PPK2)为研究目标，利用pET-29a构建重组质粒，在大肠杆菌(Escherichia coli)BL21(DE3)中表达，并将其作为ATP再生系统的关键酶与l-氨基酸连接酶(YwfE)联用生产丙谷二肽(Ala-Gln)。ppk2长度为810bp，编码270个氨基酸；SDS-PAGE结果表明PPK2为可溶性表达，分子量为29.7kDa。对PPK2的最适反应条件进行了优化，结果发现其在22–42℃、pH7–10的范围内均可以保持较好活性，且在37℃、pH为7、镁离子(Mg²⁺)浓度为30mmol/L、底物ADP与六偏磷酸钠浓度分别为5mmol/L和10mmol/L时酶活最大，在0.5h时ATP产率可以达到理论值的60%以上。作为模式反应体系，当PPK2与YwfE联用生产Ala-Gln时，达到与直接添加ATP相同的效果。此聚磷酸激酶作为ATP再生系统具有较好的适用性，适用的温度和pH范围广，且能以廉价易得的短链polyP为底物高效合成ATP，为依赖ATP的催化反应体系的能量再生提供了新酶的来源。

Abstract:

Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S.siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42℃ and pH 7-10. The highest enzyme activity was observed at 37℃, pH 7, 30 mmol/L magnesium ion (Mg²⁺), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S.siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.