Cloning And Expression Of 5-aminolevulinate Dehydratase From Escherichia Coli Rosetta(DE3) And Primary Study On Its Enzymatic Properties

5-aminolevulinic acid(ALA)is the first common precursor in the biosynthesis of tetrapyrrole compounds such as heme,porphyrins,chlorophyll and vitamin B₁₂, and exists widely in the cells of microorganisms,plants and animals.Recently,it has been found that ALA can be used as green herbicide and bio-pesticide as well as a new generation of photodynamic medicine,which has potential application for the treatments of cancers,skin diseases and lead poisoning diagnosis.A recombinant Escherichia coli for ALA production has been successfully constructed via the C4 pathway in microorganism.The over expressed ALA synthase catalyzed the condensation between succinyl-CoA and glycine.The highest ALA productivity reached 6.6 g/L(50 mM).5- aminolevulinate dehydratase(ALAD,or porphobilinogen synthase,PBGS, E.C.4.2.1.24)catalyzes the condensation of two molecules of ALA to one molecule of porphobilinogen.As the downstream enzyme of ALA biosynthesis,ALAD inevitably reduces the ALA productivity in the fermentation of recombinant E.coli, however,the absence of ALAD will block normal metabolizability of the cells. Studing on the enzymatic reaction dynamic properties of ALAD will benefit to find a certain methods to inhibit the activity of ALAD,and thus provides the possibility of enhancement of ALA productivity.In this work,gene of ALAD was cloned from E.coli Rosseta(DE3)and ligated into plasmid vector pET-28a(+).The resulting recombinant plasmid was named as pET28a(+)-hemB.After transformation the recombinant plasmid into the E.coli host strain BL21(DE3),a single colony carrying the recombinant plasmid was picked out and induced to yield recombinant ALAD using 1 mM IPTG.The recombinant ALAD, with fusion tag,migrated at 36.5 kD in SDS-PAGE.ALAD activity was assayed by the modified Ehrlich reagent as 3.10×10³ U/mg protein.The induction conditions of recombinant ALAD was optimized as following: 0.4 mM IPTG was added at 2 h after fermentation beginning,then changed the temperature of culture to 28℃and cultivated another 4 h.After affinity chromatography,gel filtration chromatography and freeze drying,purified ALAD was obtained and the specific activity was improved by 6.19 times.In addition,properties of ALAD expressed by recombinant E.coli were studied. The optimal reaction pH and temperature of the recombinant ALAD are pH 7.5 and 37℃,respectiviely.K_m of the ALAD is 0.569 mM.Mn²⁺,Ca²⁺and K⁺ activated ALAD markedly,Cu²⁺and Li⁺ inhibited ALAD markedly,10 mM EDTA inactivated ALAD by 90%,and Mg²⁺made a big contribution to ALAD activity.Dithiothreitol at low concentration increased ALAD activity.ALAD activity was significantly inhibited by substrate analog levulinic acid,D-glucose and D-xylose.