Expression, Purification, And Characterization Of Arginine Deiminase From Lactobacillus Brevis CGMCC 1306 In Escherichia Coli BL21

Arginine deiminase (ADI, EC 3.5.3.6) is a kind of arginine degradation enzyme, and regarded as a promising novel drug for arginine-auxotrophic tumors, such as hepatocellular carcinoma (HCC) and melanoma. In addition, ADI is more effective for acute lymphoblastic leukemia than L-asparagine. However, all of the ADIs applied in clinical studies are originated from Mycoplasma arginini, leaving certain safety risks. Moreover, there are several common limitations such as low enzyme activity, short half-life and low substrate affinity among ADIs reported so far. Therefore, it is of great significance to develop a more effective and safe source for ADI.In this study, an arginine-dihydrolase-positive probiotics Lactobacillus brevis CGMCC 1306 was used as the material, its arginine pathway characteristics and arginine hydrolysis gene cluster were analyzed firstly, then its ADI encoding gene was cloned for the first time, and over-expressed in E. coli BL21 (DE3). The enzymatic properties of recombinant ADI were also studied. The main results are as follows:The chromatography analysis of arginine hydrolyzate showed that the arginine hydrolysis system of L. brevis CGMCC 1306 was intact, thus it could degrade arginine into citrulline, ornithine and generate ATP, CO2, ammonia by ADI, ornithine carbamoyl transferase (OCT) and carbamate kinase (CK) in slightly acidic environment. The gene cluster of arginine pathway was located in the chromosome genome of L. brevis CGMCC 1306 with a structure of arcABDTCR, which was coincident with its arginine pathway characteristics but of great difference with other microorganism reported so far.The arc A gene of L. brevis CGMCC 1306 was acquired using PCR amplification, sequence analysis showed a fragment of 1233 bp, encoding 410 amino acids, and the calculating protein molecular weight was 45.9 kDa. Through the Blast analysis of NCBI, it only showed difference at one single nucleotide with arcA gene of Lactobacillus brevis KB290, their amino acid sequences were identical. Compared with ADIs from other Lactic acid bacteria (LAB), such as Lactobacillus oryzae JCM 18671 and Lactobacillus buchneri NRRL B-30929, the protein identity was up to 91% and 82%, respectively. Though its amino acid sequence shows only 34%similarity with other non-LABs, such as Pseudomonas aeruginosa and Mycoplasma arginini, they still share the same conserved sequence (FTRD, EGGD, MHLDT, CMSxP) and catalytic triad (C-H-G).By using pET-28a(+), the arcA gene was over-expressed in E. coli BL21 (DE3). However, we found that the recombinant ADI was highly sensitive to imidazole, it was deactivated after Ni-NTA affinity chromatography and its activity could not be restored after dialysis. Thus E. coli BL21 (DE3)/pET-21a-ADI was constructed to acquire non-redundant ADI, the purified enzyme was acquired through HiPrep DEAE FF ion exchange chromatography and SeperdexTM G-200 gel filtration.2.8 mg rADI could be obtained from per liter fermentation broth, which was purified up to 55 times with specific activity of 71.5 U/mg. The molecular weight of rADI in the native condition was 183 kDa, constructed by homotetramer. The optimum temperature and pH for rADI were 60℃ and 6.0, its half-life at 60℃ was only 10 min, and the enzyme activity dropped to undetectable level after being treated at 60℃ for 40 min. The Km and Vmax values for arginine were 1.79 mM and 1.69 μmol/s-mL, respectively.