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Appl Biochem Biotechnol. 2013 Apr;169(8):2442-56. doi: 10.1007/s12010-013-0136-z. Epub 2013 Mar 2.

Gene cloning, expression, and characterization of a cyclic nucleotide phosphodiesterase from Arthrobacter sp. CGMCC 3584.

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  • 1College of Life Science and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, 210009, People's Republic of China.

Abstract

Based on thermal asymmetric interlaced polymerase chain reaction, the arpde gene encoding a cyclic nucleotide-specific phosphodiesterase was cloned from Arthrobacter sp. CGMCC 3584 for the first time. The 930-bp region encoded a 309-amino-acid protein with a molecular weight of 33.6 kDa. The recombinant ArPDE was able to hydrolyze 3',5'-cAMP, 3',5'-cGMP, and 2',3'-cAMP. The K m values of ArPDE for 3',5'-cAMP and 3',5'-cGMP were 6.82 and 12.82 mM, respectively. ArPDE was thermostable and displayed optimal activity at 45 °C and pH 7.5. The enzyme did not require any metal cofactors, although its activity was stimulated by 2 mM Co(2+) and inhibited by Zn(2+). Nucleotides, reducing agents, and sulfhydryl reagents had different inhibitory effects on the activity of ArPDE. NaF, the actual compound used to improve the industrial yield of cAMP, exhibited 62 % inhibitions at concentrations of 10 mM.

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