1887

Abstract

The gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extremely thermophilic bacterium, , was directly sequenced by the thermal asymmetric interlaced (TAIL) PCR method. An ORF for a 937 amino acid polypeptide was found in the gene. The gene had a high G+C content (66.2 mol%) and the third position of the codon exhibited strong preference for G or C usage (85.0 mol%). The calculated molecular mass was 107 848 Da, which was consistent with the molecular mass of the enzyme as determined by SDS-PAGE (100 kDa). The amino acid sequence of PEPC was closely related to that of PEPC from another thermophile, a sp., and from a mesophile. , exhibiting 45.3% or 37.7% identity and 61.5% or 56.5% similarity, respectively. By Southern analysis, the gene was found to be present in a single copy in the genomic DNA of this organism. The cloned gene was expressed in using a pET expression vector system and a thermostable recombinant PEPC was obtained. Comparison of the deduced amino acid sequences of the thermophilic and mesophilic PEPCs revealed distinct or common preferences for specific amino acid composition and substitutions in the two thermophilic enzymes.

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1998-05-01
2022-01-17
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