Enzymatic Production Of Agmatine From L-arginine

Agmatine,an important neurotransmitter,plays vital roles in L-arginine-NO pathway and biogenic amine metabolic and is widely used in neurotransmitter systems,nitric oxide synthesis,and polyamine metabolism,and also used as functional foods and additives.In this study,we started from the perspective of decarboxylation of L-arginine.After genome mining and functional screening,an L-arginine decarboxylase(ADC,EC 4.1.1.19),named SpA9 with high catalytic efficiency from Shewanella putrefaciens was selected.Investigations on SpA9 enzymatic properties were characterized,and a high efficiency transformation condition for agmatine production was also established.1.Screening and expression of ADC in E.coli BL21.Based on the chemical structure of L-arginine,ADC coded with speA gene from E.coli was chosen as the probe sequence,and a decarboxylase toolbox was developed using a genome-mining strategy.Seven different ADCs with certainly identity with the probe sequence were selected.A particular type of ADC named SpA9 from S.putrefaciens exhibited the highest enzyme activity with 15.8 U?mg-1,which is 29.8 times more than the probe protein.2.Purification and characterization of SpA9.The specific activity of purified SpA9 was found to be 121.9 U?mg-1 with a mocelular weight of 70.8 kDa.The optimal temperature of SpA9 was 37°C.The enzyme activity decreased with increasing temperature after 37°C.Furthermore,SpA9 lost 61% of its relative activity after 1-h incubation at the optimum temperature(37°C).The maximum activity of SpA9 was observed at pH 8.5,and poor activity was observed at pH <8.0 or >9.0.SpA9 kept 94% of initial activity after incubation for 1 hour at pH 8.5.The effect of metal ions on SpA9 activity was also investigated(Mg2+ used as the control).Zn2+,Mn2+,Ca2+,Fe2+,Cu2+,and K+ inhibited enzyme activity by 18.2%,37.1%,48.3%,42.5%,23.1%,and 45.7%,respectively.SpA9 yielded a Km value of 2.2 mmol?L-1 and a Kcat value of 10.4 s-1,resulting in a catalytic efficiency(Kcat/Km)of 4.8 mM-1? s-1.The specific activity of SpA9 towards 15 common amino acids(chain substitution)was evaluated,and only L-arginine was catalysed.An H-bond was only found in SpA9-Arg complex with Cys507.3.Establishment and optimization of transformation condition.The optimised induction medium includes 18 g?L-1 tryptone,5 g?L-1 yeast extract,4.6 g?L-1 glucose,0.5 g?L-1 NaCl,0.186 g?L-1 KCl,and 2.033 g?L-1 MgCl2·6H2O.The highest enzyme activity(1281 U?mL-1)was achieved when the recombinant E.coli BL21/pET28a-SpA9 strain was induced at 25°C with 7 g?L-1 lactose for 12 h.The optimum transformation systems was also investigated.Ennhanced condition includes 90 g?L-1 l-arginine,31.5 mmol?L-1 PLP,18 mmol?L-1 MgSO4,2% Triton-100 and 135 g?L-1 whole-cell biocatalysts(E.coli BL21/pET28a-SpA9).The reaction was catalysed in a 3.5-L fermenter at 37°C,pH 8.5,400 rpm,and 1.0 vvm.The highest agmatine production concentration reached 52.66 g?L-1,with a conversion rate of 78.27% and STY of 26.33 g?L-1?h-1.