Recombinant Expression, Fermentation Optimization And Application Of Arginine Deiminase From Pseudomonas Putida

Arginine deiminase(ADI, EC 3.5.3.6) can catalyze L-arginine or L-arginine hydrochloride, generating L-citrulline and ammonia. L-citrulline is one kind of non-protein amino acid. It plays an important role in the in vivo metabolism of ammonia. Meanwhile it has broad application prospects in medical supplies, cosmetics, health care product and food additives, etc. ADI from Pseudomonas putida has become a hot topic because of its good stability and high biotransformation rate to L-citrulline, etc. But it is intracellular localization protein and usually expressed intracellularly. In order to solve the problem of extracellular expression of these proteins, our preliminary study shows that T. fusca cutinase can hydrolysis membrane phospholipid components restrictedly to enhance membrane permeability, then intracellular protein achieves nonspecific leakage without causing cell lysis.In this study, the gene of ADI from Pseudomonas putida ACCC 10185 was cloned and expressed in Escherichia coli BL21(DE3)(E. coli BL21(DE3)). We carried out enzymatic property analysis and catalytic process research for L-citrulline preparation of recombinase obtained by two expression ways. High density fermentation conditions of co-expression recombinant strain was optimized to enhance the level of extracellular ADI expression. The main results were listed as follows:(1) ADI was expressed successfully in E. coli. The full length of 1263 bp of gene arc A was amplified by PCR with P. putida ACCC 10185 genome as a template. Gene arc A was connected with expression vector p ET-24a(+) or p ETDuet-1-cutopt. Then genetically engineered strains were constructed of E. coli BL21(DE3)/p ET-24a(+)-arc A(ADI produced was ADID) and E. coli BL21(DE3)/p ETDuet-1-cutopt-arc A(ADI produced was ADIG). Shake flask fermentation showed that when ADI expressed separately, the recombinant strain was cultured in TB medium and the ADID activity(0.7 U×mg-1 wet cells) on the intracellular supernatant was reached after cultivation at 25°C for 18 h. When ADI was expressed with cutinase of optimized gene, the recombinant strain was cultured in TB medium and the ADIG activity(7.7 U×m L-1) on the culture supernatant was reached after cultivation at 25°C for 34 h which accounted 90.9% for the total activity. ADI of intracellular localization was extracellularly expressed successfully.(2) Enzymatic properties were studied of recombinase. Studies have shown that the optimum temperature of ADID and ADIG was all 37°C. Half amount of recombinant ADI at 37°C was all 15 h. The optimum p H of ADID and ADIG was all 6.0. Recombinant ADI was stable at p H 6.5-7.5. Common metal ions had no influence on the recombinase enzyme activity. Subsequently, Kinetics of enzymatic reactions of ADID and ADIG was performed under the conditions of 37°C, p H 6.0 and L-arginine hydrochloride as substrate. Michaelis constant(Km) and the maximum reaction rate(Vmax) of ADID were 0.21 mmol×L-1 and 107.3 mmol×L-1×min-1×mg-1. Km and Vmax of ADIG were 0.23 mmol×L-1 and 106.9 mmol×L-1×min-1× mg-1.(3) The process was optimized for preparing L-citrulline by recombinase. Several reaction conditions were studied and optimized with L-arginine hydrochloride as substrate, including the initial reaction p H, reaction temperature, enzyme dosage, speed, L-arginine hydrochloride concentration and reaction time. The optimal process was all that initial reaction p H 6.0, reaction temperature 37°C, L-arginine hydrochloride 100 g×L-1, ADI 24 U×g-1 substrate, 100-200 r×min-1 and the conversion rate could achieved 100% after 2.5 h. When the concentration of L-arginine hydrochloride was increased to 650 g×L-1, the conversion rate of L-citrulline can still achieved 100%.(4) 3 L tank high density fermentation conditions were optimized of extracellular expression ADI in recombinant strain. Recombinant strain of co-expression utilized the induction strategy of 25.0 μmol×L-1 IPTG one-time added and 0.1 g×L-1×h-1 lactose fed at constant speed after optimization with induction temperature and lactose inducing concentration. Enzyme activity of ADIG on the culture supernatant reached 101.2 U×m L-1 which accounted 95% for the total activity. It was the highest ADI activity reported.