Heterologous Expression Of Arginine Deiminase In Photosynthetic Bacteria And Biosynthesis Of L-citrulline

Arginine deiminase（ADI）,which is capable of hydrolyzing L-arginine to generate L-citrulline and ammonia.L-citrulline plays an important role in ammonia metabolism in living organisms and has promising prospects in medicine,cosmeceuticals as well as food.In this study,the gene of interest,Arca ligated expression vector p ET28a,was transformed into the expression host E.coli BL21 by ADI.To further validate its ability to be expressed in a photosynthetic bacterial expression system,the selected photosynthetic bacterium was R.rubrum H2.First,the p PUCTerm-arc A expression vector was constructed by gene recombination,and R.rubrum H2 was used as the expression host of membrane protein.The vector ppucterm Arca was transformed into R.rubrum H2 according to the special transformation ability of E.coli WM3064,and ADI was checked for no expression by SDS-PAGE assay,We also studied the effect of three conditions,temperature,p H as well as metal ions,on the activity of ADI.In order to further simplify the production process,this experiment explored the optimal conditions for bioconversion of L-citrulline by recombinant bacteria,and determined the optimal reaction p H,temperature,substrate concentration,buffer concentration,and other conditions.The results showed that the recombinant plasmid p ET28a-arc A was identified by PCR,two enzyme digestion validation method and sequence alignment,and p PUCTerm-arc A was successfully constructed;SDS-PAGE was used to identify the presence of ADI in E.coli BL21 in the form of inclusion bodies,and the protein concentration determined by Coomassie brilliant blue method was 0.385 mg/m L.Highly expressed in the photosynthetic bacteria R.rubrum H2 expression system,the polyacrylamide gel electrophoresis results showed that the target protein was successfully expressed and consistent with the theoretical molecular weight of 46 k Da.The target gene exists as a membrane protein in R.rubrum H2.The specific activity of the purified recombinant enzyme is 10.02 U/mg.The results of the study on the characteristics of the recombinant enzyme showed that the optimum p H of the recombinant enzyme was 6,the optimum temperature was 40℃,and Mn²⁺could improve the activity of the enzyme,while Cu²⁺and Zn²⁺inhibited the catalytic activity of the enzyme.Through optimizing the transformation conditions of L-citrulline by recombinant enzyme,the results showed that the best transformation efficiency was achieved when the optimal transformation temperature was 40℃,the optimal transformation p H was 6.5,the addition concentration of L-arginine was 100 g·L^-1,and the buffer concentration was 0.02 mol·L^-1.The above results can provide theoretical data for subsequent studies on heterologous expression of arginine deiminase and preparation of L-citrulline.