Identification And Directed Evolution Of Arginine Deiminase From Pseudomonas Plecoglossicida

Arginine deiminase (ADI; EC 3.5.3.6) belongs to a guanidine group-modifying enzyme superfamily and catalyzes the irreversible hydrolysis of arginine to citrulline and ammonia, is expressed in microorganisms, but not in mammalian cells. It has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas (HCCs).HCC is one of the most common malignancies in the world, and its annual incidence is approximately 1 million cases. In 1999 and 2005, ADI-PEG-20, PEGylated recombinant ADI was designated the US orphan drug status for the treatment of HCC and invasive malignant melanomas by FDA and European Agency for the Evaluation of Medicinal Products (EMEA). Currently, clinical trials of ADI-PEG-20 for the treatment of HCCs (Phase III) and melanomas (Phase I/II) are being investigated.In this study, a strain SWP1 producing high activity of ADI was isolated from the Wuxi canal. Based on its morphological, biochemical characteristics and 16S rRNA gene sequence analysis, SWP1 was identified as Pseudomonas plecoglossicida and is now deposited at CGMCC (China General Microbiological Culture Collection Center) as P. plecoglossicida CGMCC2039. In addition, a Chinese patent (An arginine deiminase producing strain and its application, Application No.200710107822.X) was applied.Recombinant vector pET 24a-ADI was constructed for the over-expressed of ADI in E. coli BL21(DE3). The effects of IPTG concentrations, induction temperatures and induction time were investigated. Under the optimal expression conditions the enzyme activity reached to 2.04 U/mL broth. The rADI was purified using DEAE Fast Flow anion exchange and Superdex 200 gel filtration column chromatography. The rADI had a molecular mass of about 92.6 kDa, and comprised a homodimer of 46 kDa in the native condition. The specific activity of rADI was up to 20.85 U/mg. The Km and Vmax values for arginine were 2.88 mmol/L and 31.68μmol/min/mg, respectively, and the optimum temperature and pH for the production of rADI were 40℃and 6.0.It was noticed that the optimum pH of ADI from Pseudomonas plecoglossicida (PpADI) was 6.0, and less than 10%of the activity was retained at pH 7.4 (pH of human plasma is around 7.4). Additionally, the Km value for wild type ADI (WT-ADI) was 2.88 mmol/L (pH 6.0), which is over 20 times of the serum arginine level (100-120μM). These are two major limitations for PpADI as a potential anti-cancer drug. Directed evolution has proven to be a powerful tool to modify a biological molecule towards a desirable property. A highly sensitive and efficient high throughput screening strategy employing ep-PCR and a modified diacetylmonoxime-thiosemicarbazide (DAM-TSC) method was established to isolate ADI mutants with higher activity and lower Km under physiological pH. One excellent mutant 314 (M314, A128T, H404R, I410L) was selected among 650 variants after one round of ep-PCR and screening. The specific activity of M314 (9.02 U/mg) is over 20-fold higher than that of WT-ADI (0.44 U/mg) at pH 7.4, and the Km value was reduced to 0.65 mM (pH 7.4). Noticeably, the pH optimum was shifted from 6.0 to 6.5 in M314. Homology model of M314 was constructed to understand the molecular basis of the improved enzymatic properties.