Molecular cloning, overproduction and characterization of the Bacillus cereus IMP dehydrogenase

The gene of IMP dehydrogenase of Bacillus cereus ts-4, a temperature-sensit ive mutant of B. cereus JCM 2152, was subcloned and its sequence was analyz ed. A B. cereus ts-4 DNA fragment of 2,065 bp containing the entire impdh g ene and flanking regions was sequenced. The fragment contained an open read ing frame of 1,527 bp encoding 509 amino acids with a calculated molecular mass of 55,390 Da. The impdh sequence of JCM 2152 was also analyzed by TA c loning using PCR products amplified with primers from B. cereus ts-4 impdh gene. The gene amplified by PCR was expressed in Escherichia coil using a p ET17 x b expression plasmid. The N-terminal amino acid sequence of the over produced enzyme was identified as Met-Trp-Glu-Ser-Lys-Phe-Val-Lys-Glu-Gly-L eu-Thr-Phe-Asp-Asp-Val-Leu-Leu-Val-Pro. The overproduced enzyme was eluted at a molecular mass of about 225 kDa by gel filtration. The molecular mass of the subunit was estimated to be 56 kDa by SDS-PAGE. The overproduced enz yme was active against IMP, IDP, and ITP, and showed the highest activity a t pH 9.5. These properties of the recombinant enzyme were almost identical to those of LMP dehydrogenase of B. cereus.