Abstract: Adenylate cyclase (AC) is the key enzyme that catalyzes the formation of cAMP from ATP. In this study, we discovered two novel class III ACs with a halophilic property from Thermobifida halotolerans DSM 44931 (ThAC) and Haloactinopolyspora alba DSM 45211 (HaAC), respectively. The recombinant ThAC and HaAC were expressed in Escherichia coli with molecular weights of 36.1 and 36.0 kDa respectively. The presence of 2500 and 2200 mmol·L^-1 NaCl significantly enhanced the enzyme activities of ThAC and HaAC, with 22-fold and 7.4-fold higher activities compared to those without NaCl, respectively. Several divalent metal ions were found to activate the recombinant ACs to different extents, and the optimal metal ion was Mg²⁺ for both ThAC and HaAC with concentrations of 80 mmol·L^-1 and 40 mmol·L^-1 respectively. Purified ThAC and HaAC had the optimal specific activities ((4.59 ±0.35)×10⁴ and (7.76 ±0.52)×10⁴ U·mg^-1) and catalytic efficiency (4.47 and 5.30 L·mmol^-1·s^-1) at 45 ℃ and 40 ℃ respectively, while the optimum pH of both two recombinant ACs was 10.0. This is the first report of the halophilic Class III ACs, which could make new contributions to explore and study ACs for further associated investigations.

Key words: Enzyme, Protein stability, Biotechnology, Halophilic adenylate cyclase, cAMP

摘要： Adenylate cyclase (AC) is the key enzyme that catalyzes the formation of cAMP from ATP. In this study, we discovered two novel class III ACs with a halophilic property from Thermobifida halotolerans DSM 44931 (ThAC) and Haloactinopolyspora alba DSM 45211 (HaAC), respectively. The recombinant ThAC and HaAC were expressed in Escherichia coli with molecular weights of 36.1 and 36.0 kDa respectively. The presence of 2500 and 2200 mmol·L^-1 NaCl significantly enhanced the enzyme activities of ThAC and HaAC, with 22-fold and 7.4-fold higher activities compared to those without NaCl, respectively. Several divalent metal ions were found to activate the recombinant ACs to different extents, and the optimal metal ion was Mg²⁺ for both ThAC and HaAC with concentrations of 80 mmol·L^-1 and 40 mmol·L^-1 respectively. Purified ThAC and HaAC had the optimal specific activities ((4.59 ±0.35)×10⁴ and (7.76 ±0.52)×10⁴ U·mg^-1) and catalytic efficiency (4.47 and 5.30 L·mmol^-1·s^-1) at 45 ℃ and 40 ℃ respectively, while the optimum pH of both two recombinant ACs was 10.0. This is the first report of the halophilic Class III ACs, which could make new contributions to explore and study ACs for further associated investigations.

关键词: Enzyme, Protein stability, Biotechnology, Halophilic adenylate cyclase, cAMP