title: High activity and stability of codon-optimized phosphoenolpyruvate carboxylase from Photobacterium profundum SS9 at low temperatures and its application for in vitro production of oxaloacetate.
abstract: Phosphoenolpyruvate carboxylase (PEPC) of Photobacterium profundum SS9 can be expressed and purified using the Escherichia coli expression system. In this study, a codon-optimized PEPC gene (OPPP) was used to increase expression levels. We confirmed OPPP expression and purified it from extracts of recombinant E. coli SGJS117 harboring the OPPP gene. The purified OPPP showed a specific activity value of 80.3 U/mg protein. The OPPP was stable under low temperature (5-30 °C) and weakly basic conditions (pH 8.5-10). The enzymatic ability of OPPP was investigated for in vitro production of oxaloacetate using phosphoenolpyruvate (PEP) and bicarbonate. Only samples containing the OPPP, PEP, and bicarbonate resulted in oxaloacetate production. OPPP production system using E. coli could be a platform technology to produce high yields of heterogeneous gene and provide the PEPC enzyme, which has high enzyme activity.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, 121-742, Republic of Korea.
authors.forename: Soohyun
authors.initials: S
authors.lastname: Park
authors.forename: Soohye
authors.initials: S
authors.lastname: Hong
authors.forename: Seung Pil
authors.initials: SP
authors.lastname: Pack
authors.forename: Jinwon
authors.initials: J
authors.lastname: Lee
mesh_terms: D001483:Base Sequence; D003062:Codon; D003080:Cold Temperature; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D010071:Oxaloacetates / Q000378:metabolism*; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010776:Photobacterium / Q000201:enzymology*
chemical_list: D003062:Codon; D017931:DNA Primers; D010071:Oxaloacetates; D010730:Phosphoenolpyruvate Carboxylase