Enzymatic Properties Of L-Lysine Decarboxylase And Its Application In Cadaverine Production

Lysine decarboxylase(LDC)is the key enzyme of cadaverine biosynthesis pathway,catalyze the conversion of L-lysine to cadaverine and carbon dioxide.The catalytic product cadaverine widely exists in animals,plants and microorganisms.It is a bioactive nitrogenous alkaline organic compound,has extensive application in industry for synthesis of high performance nylon material.Therefore,a good LDC having high catalytie activity,high pH stability and thermal stability is of great importance for the development of industrial production and application of cadaverine.In the present thesis,24 LDC candidates from different sources,together with two equivalents E.coli CadA and LdeC as control,were selected by using database enzyme mining method.Cloning of these enzymes were earried out in E.coli,and 10 LDCs were successfully and stably expressed by induction optimization.Among them,three enzymes from Photobacterium angustum,Aliivibrio salmonicida,Edwardsiella tarda,abbreviated as PaLdc,AsLdc and EtLdc,respectively,were found to exhibit higher enzyme eatalytic activity to L-lysine,and thus were further purified for characterization.The optimum pH of CadA,LdcC,PaLdc,AsLdc and EtLdc are 5.5,7.5,6.5,7.5,7.0,and the maximum enzyme activity are 171.6,51.5,176.3,205.1,141.9U·mg-1,respectively.Under alkaline condition such as pH 8.5,AsLdc and EtLdc still showed residual relative enzyme activities of 45.6%and 42.5%,respectively,whereas CadA,LdcC and PaLdc reduced to 7%.26.8%and 5.2%of their relative activities,respectively.The optimum temperatures of CadA,LdcC,PaLdc,AsLdc and EtLdc under each optimum pH condition were 50?,55?,60?,50? and 60?,respectively.After incubated at 50? for 12 hours,CadA,LdcC,PaLdc,AsLdc and EtLdc kept 50.8%,0%,61.7%,75.2%and 46.3%of relative activity,respectively,and this indicating the LdcC is thermal labile.Km values of L-lysine of the five enzyms were 0.75 mM,1.24 mM,1.15 mM,0.94 mM and 0.68 mM,respectively.Inhibition effect of different cadaverine concentrations on LDCs were determined and found that about 4-6 mM cadaverine inhibited 50%of their activity.Moreover,the relationship among oligomeric state of the LDCs,pH value and the activity was studied by using native PAGE electrophoresis and dynamic light scattering method.The highest proportion of AsLdc decamer was observed at pH 7.5,which agrees well with the fact that the highest activity of AsLdc appears at pH 7.5.Similarly,the highest percentage of CadA decamer was observed at its optimum pH of 5.5.Therefore,the decamer state is likely the most active form of AsLdc and CadA.The results suggested that pH may contribute to the stabilization of oligomeric states of LDCs and further affect their activities.Finally,whole-cell catalysis with the novel enzymes AsLdc and CadA was carried out in a 1L fermentor.AsLdc and CadA showed similar catalytic efficiency at pH 5.5 after 7 hours reaction,with substrate conversion efficiency at 91.4%and 92.6%,and cadaverine molar yield at 98.6%and 98.5%,respectively.AsLdc showed a better catalytic ability at more alkaline pH such as pH 7.5,with substrate conversion efficiency at 100%and 82.8%,and cadaverine molar yield 96.9%and 97.5%,after 7 hours reaction.The results indicated that AsLdc has an obvious advantage for catalysis under alkaline condition,which would significantly reduce the acid usage in industrial catalytic production of cadaverine,therefore reduce the negative influence on environment.