Fermentative Production Of Arginine Deiminase By Pseudomonas Sp. And Its Activity Study

Arginine deiminase (ADI) pathway could produce one molecular of ATP by degrading one molecular of arginine, which is a major energy source of some microorganisms. ADI catalyzes the deamination of arginine to citrulline and ammonia, the first step of ADI pathway. Arginine is a nonessential amino acid for human beings since it could be produced by argininosuccinate synthetase (ASS) in normal cells and tissues. Some human cancer cell lines do not express ASS, and are therefore auxotrophic for arginine. Consequently, ADI could be used for the control or extinct of arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas (HCCs).In previous studies, ADI from Mycoplasma has been used in the clinical trials, which has potential biological safety issues. Pseudomonas sp. isolated by our laboratory has been used for ADI production. The ADI production by Pseudomonas sp. was increased for 35% from 1.34 U/mL broth to 1.82 U/mL broth after 24 h of growth in the optimized medium (g/L, glucose 10, yeast extract 5, peptone 4, arg-HCl 8, pH 7.0) with 4% inoculation, 60/250 mL capacity, and 190 rpm agitation under 30℃. The cultivation in a 5 L bioreactor showed a better result of 2.17 U/mL broth (57% increase).The ADI was purified through several steps including salting out by ammonium sulphate, dialysis, ion-exchange chromatography by DEAE Sephadex A-50, and gel filtration chromatography by Sephadex G-75. After the purification, a single band at about 54 kDa was observed on SDS-PAGE gel. After the purification, the overall activity yield of ADI is 5.8%, purification factor is 11.0, and the specific reactivity of ADI is 5.28 U/mg.Preliminary studies showed that ADI had anti-cancer activity both in vivo and in vitro. The inhibition rate to HepG2 cells by crude ADI was 93.4% at 0.5 U/mL. After the purification, the inhibition rate was 23.4% under an activity of 0.03 U/mL. In a two-week in vivo anti-cancer activity experiment, the inhibition rate to H22 cell line by crude ADI reached 83.4% at an 5 U-treatment, together with a minimal negative effect on immune organs. The results demonstrate that ADI could be applied as a potential anti-cancer drug.