Preparation And Application Of Recombinant Escherichia Coli L-aspartate-a-decdarboxylase

The major route of B-alanine production for CoA in bacteria is by the a-decdarboxylation of L-aspartate, catalyzed by the L-aspartate-a-decdarboxylase (EC4.1.1.11, ADC). Recombinant ADC was expressed in E. coli successfully. The feasibility of production of B-alanine using recombinant ADC to catalyze L-aspartate was investigated in this paper.The Escherichia coli gene encoding L-aspartate-a-decdarboxylase was cloned by PCR and ligated with the vector pET-28a(+) to get the expression plasmid panDpET. The panDpET plasmid was transformed into E. coli BL21(DE3) cells by heat shock procedure for overproduction of the recombinant ADC.A new method for ADC activity assay was established by evaluation the conversion of the aspartate to β-alanine as determined by reverse phase high performance liquid chromatography (RP-HPLC). L-aspartate and β-alanine were derived by o-Phthalaldehyde (OPA) method. Isocratic column eluton was carried out at1.00mL/min using20mM sodium acetate pH5.9containing25%(v/v) acetonitrile., detected at340nm. The retention time was1.5min for aspartate,3.5min for B-alanine and6.5min for OPA respectively.The formation of active ADC was studied by incubating recombinant zymogen at varying temperature and pH. The zymogen were activated completely incubated at37℃, pH8.0for22hours.The production of B-alanine from aspartate catalyzed by recombinant ADC was studied. The reaction condition was determined, as at25℃, pH7.0. The rate of conversion was about12μM aspartate with1μg ADC. The recombinant ADC was stable while kept under15℃to37℃in pH6.0to pH9.0buffer.