[Expression of polyphosphate kinase from and its application in ATP regeneration system].

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.