Identification Of L-aspertate Decarboxylase Encoding Genes And Analysis Of Enzyme Function

L-aspartate ?-decarboxylase(PanD),an important regulation enzyme in the pantothenic acid synthesis pathway,could specifically catalyze the decarboxylation of L-aspartate at the ?-position to produce ?-alanine with high value.Enzymantic synthesis of ?-alanine has great potential for industrialization.However,there were still some problems that prevent the industrial application of the PanD enzyme,such as limited gene sources of the recombinant PanDs,unclear self-cleavage mechanism,and substrate inactivation on all of recombinant PanDs that have been reported.In this paper,panDs genes from different sources were expressed in Escherichia coli.The self-cleavage,catalytic properties and the rate of substrate inactivation of the purified recombinant enzymes were studied.44 optimized panD genes were expressed in Escherichia coli,respectively.The recombinant enzymes were purified by HisTrapTM affinity chromatography,and the catalytic properties of them were characterized.Compared with PanDs from C.glutamicum and B.subtilis,there were also 6 recombinant PanDs with a higher specific activity.Posttranslational self-cleavage of the recombinant PanDs in this study could be classed three class: almost no cleavage,partial cleavage and complete cleavage.The self-cleavage results of PanD maybe indicate the homologues of these enzymens.Incubation could promote PanD zymogen cleavage,but the catalytic activity increasing was not significant.In this research,a remarkably substrate inactivation occurs during catalysis,probably caused by irreversible transamination of the catalytically essential pyruvoyl group.The rate of inactivation was dependent on the concentration of substrate,and the residual activity assumed a pseudo-first-order reaction at the same condition.Exspecially,We found PanD from Corynebacterium jeikeium showed a highest specific activity up to 11.8 U·mg-1,and the PanD from L.monocytoene with a higher activity(8.9 U·mg-1),has a less substrate inhibition(2.071 × 10-3 min-1).The enzymatic properties and capability of bioconversion of them were studied.PanDL.m and PanDC.j exhibited the same optimal temperature at 60?,while they possessed different optimal pH at 7.0 and 6.0,respectively.Both the enzymes were stable at the condition of 30-50?,and pH 4.0-7.0.Under the same conditions,the conversion rates of L-aspartate were 67.7% and 82.2%,respectively,with the potential for industrial applications.