Huang Xin, Li Yimin, DU Cong, Yuan Wenjie
School of Bioengineering, Dalian University of Technology, Dalian 116024, Liaoning, China.
Ningbo Research Institute, Dalian University of Technology, Ningbo 315000, Zhejiang, China.
Sheng Wu Gong Cheng Xue Bao. 2022 Dec 25;38(12):4669-4680. doi: 10.13345/j.cjb.220320.
Polyphosphate kinase plays an important role in the catalytic synthesis of ATP . 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 was cloned and expressed in 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 of . 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 (Mg), 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 . 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.
多磷酸激酶在ATP的催化合成中起重要作用。为了找到一种能以短链多聚磷酸盐（polyP）为底物高效合成ATP的多磷酸激酶，克隆了来自[具体来源未给出]的多磷酸激酶2（PPK2）并在大肠杆菌BL21(DE3)中表达。作为一种ATP再生酶，PPK2与L-氨基酸连接酶（YwfE）联合使用以生产L-丙氨酰-L-谷氨酰胺（Ala-Gln）。[具体来源未给出]的PPK2长度为810 bp，编码270个氨基酸。SDS-PAGE显示PPK2正确表达，其分子量如预期为29.7 kDa。对PPK2的反应条件进行了优化。PPK2在22 - 42℃和pH 7 - 10范围内能保持良好活性。在37℃、pH 7、30 mmol/L镁离子（Mg）、5 mmol/L ADP和10 mmol/L六偏磷酸钠条件下观察到最高酶活性，在此条件下0.5小时内ATP产量达到理论值的60%。当与YwfE联合用于生产Ala-Gln时，PPK2作为ATP再生系统表现出良好的适用性，效果与直接添加ATP相似。来自[具体来源未给出]的PPK2在较宽的温度和pH范围内表现良好，以廉价且容易获得的短链polyP为底物合成ATP。因此，PPK2为依赖ATP的催化反应系统提供了一种新的酶源。
