Cloning and expression of guanosintriphosphate cyclohydrolase-I from anoxybacillus flavithermus

This study was included the characterization of guanosin triphosphate cyclohydrolase I from thermophilic bacteria, Anoxybacillus flavithermus. We cloned the gchI gene that was 603 bp and its open reading frame has 200 amino acids. This enzyme catalyzed from GTP to dihydroneopterin triphosphate. We cloned into pET28a(+) expression vector with 6xHis tags and transform in E. coli BL21(DE3)pLysS host cells to express with 1 mM IPTG induction. We purified the enzyme with Ni-NTA resin. We determined that GCH-I is 24 kDa, its optimum pH is 8.0 and temperature is 65?C. Under optimal conditions, GCH-I exhibited a low enzymatic activity with Km- and Vmax- values of 243 ± 23,25 ?M and 100,93 ± 3,5 nM/min/mg protein, respectively. Key Words: Anoxybacillus flavithermus, GTP, GTP cyclohydrolase I, enzyme characterization.