Cloning, Expression And Site-directed Mutagenesis Of A Arginine Deiminase Gene From Aeromonas Sp

L-citrulline is an important intermediate metabolites of urea cycle in human body, it is able to absorb and remove harmful free radicals and have the effect on antioxidizing. Arginine deiminase can catalyze arginine to L-citrulline. Currently, L-citrulline production methods are fermentation method, chemical method, extraction method and enzymatic method. The disadvantage of fermentation production method is the low L-citrulline yield. Products of chemical method contain D-citrulline (the optically active enantiomer of L-citrulline). Extraction process is complex relatively, besides, the purification is difficult and the cost is high as well. The advantages of enzymatic production are the mild condition and the products without type D optical enantiomers. As a kind of high efficiency and low cost of production methods, enzymatic production will play an important role in the citrulline production. In the future, arginine deiminase will play a huge role in citrulline production.In this study, a strain that has high activity of ADI was obtained by screening. Based on16Sr RNA gene sequence analysis, the strain was belong to Aeromonas sp.. The arc gene was cloned from Aeromonas sp.. The arc consists of1221bp, the initiation codon ATG, the terminator TAA. The GC content of arc is61.43%. The arc encodes406amino acid and the calculated mass of ADI is45.46kDa. The isoelectric point of ADI is6.17. Predicted by the SignalP4.1Server, the ADI has no signal peptide. According to the alignment of amino acid sequence, we found that the similarity of Pseudomonas plecoglossicida’s ADI is the highest (46%). Therefore, this research cloned a novel ADI gene from Aeromonas sp.. The arc was cloned in the vector pGEX-6P-1. Recombinant plasmid pGEX-6P-arc was constructed and expressed in Escherichia coli BL21(DE3). The recombinant ADI was induced with IPTG and the crude ADI’s conversion rate is measured, the conversion rate is30%. The ADI was purified with GST lable. Then, the enzymatic properties of purified ADI were studied. The optimum temperature and pH of ADI were50℃and6.0, respectively. The Km, Vmax, kat and kcat/Km value for arginine were0.66mmol/mL,4.37μmol.mL-1.min-1,1.15s-1and1.74mL/s.mmol, respectively. According to the corresponding references and the comprehensive analysis of arginine deiminase enzyme gene sequences, two mutation sites M128, D404R were introduced into wild-type ADI gene and obtained two mutants M128T and D404R. The mutants were expressed in E. coli BL21(DE3) and the enzymatic properties of the purified enzymes were studied. As the results show, M128T had enhanced the optimum pH from6.0to6.5, closer to the physiological pH. However, the Km and kCat value of both mutants had reduced. The activity of both mutants had declined. According to the analysis of experimental results, the two sites may reside in or near the catalytic center of an ADI. It is likely to be the conservative sites of ADI’s catalytic center. It is illustrated that the two sites play an important role in the catalysis of ADI. Specific mechanisms need combining with crystal structure analysis to explain.