Effects Of Different Systems On The Expression Of AMP Deaminase

AMP deaminase(AMP deaminase;AMPD;EC 3.5.4.6)is an aminohydrolase that efficiently catalyzes the conversion of adenosine monophosphate(AMP)to NH3 and inosinic acid(IMP),widely used in the food and medicine industries.In view of the high cost,low enzyme activity,unstable enzyme activity,difficulty in extraction and purification,etc.,the AMP deaminase gene derived from Aspergillus oryzae is cloned by molecular biology.Thereby,the high-efficiency expression of AMP deaminase in E.coli.P.pastoris and B.subtilis is realized,which provides theoretical guidance and certain technical support for realizing large-scale production in the food industry.First,in this study,the gene of AMP deaminase was obtained by PCR amplification using the Aspergillus oryzae genome as a template.The gene ORF is 2964 bp,encoding 987 amino acids,the predicted protein molecular weight is 111.25266 KDa,and the theoretical isoelectric point is 5.94.Phylogenetic tree indicated that the gene shared 99%homology with the Aspergillus oryzae RIB40.Meanwhile,To achieve high-yield expression,primers were designed to construct recombinant expression vectors pET30a-AMP,pPIC9K-AMP and pMA5-AMP and transformed into E.coli Transetta DE3,P.pastoris GS115 and B.subtilis WB600,and the corresponding recombinant strains were successfully constructed.The corresponding recombinant strain was subjected to expression verification and optimization.The results showed that the maximum enzyme activity of recombinant E.coli was 1933 U/mL,in the middle of logarithmic growth(OD600=0.5-0.8),0.5 mM of the final concentration of IPTG,and 14 h of the induction time.The optimal expression conditions of recombinant P.pastoris were as follows:high-density fermentation transfer was carried out with OD600 of 2-5,and the final concentration of methanol was added by 1%,and induced for 96 hours.Under this condition,the recombinant AMP deaminase enzyme activity was 1257 U/mL.Recombinant Bacillus subtilis can produce enzymes without adding inducer.The activity of AMP deaminase is relatively high at 8-14 h after fermentation,and the highest enzyme activity can reach 2450 U/mL,which is higher than the recombinant enzyme expressed by recombinant E.coli and recombinant P.pastoris.The effects of three expression systems on the activity and expression of AMP deaminase were compared and the B.subtilis expression host was selected as the follow-up study object.The AMP deaminase was separated and purified by Ni2+-chelating chromatography.The results showed that the 180 mM,220 mM and 260 mM imidazole buffers could elute the target protein,which was detected by ultrafiltration and SDS-PAGE as a single band to achieve electrophoretic purity.On this basisthe enzymatic properties of the recombinase were studied.The results showed that the optimal reaction temperature of the recombinant enzyme was 55?.The enzyme maintains 80.2%of the activity when treated at 35? for 30 min,and the thermal stability of the enzyme is deteriorated when the temperature is higher than 40?.The optimum pH is 6.0,and the activity is stable at pH 6.0?8.0.Zn2+,Fe3+ and Al3+ can significantly enhance the enzyme activity.Among them,the enhancement effect of Fe3+ is the most significant,and the enzyme activity can be increased by 236%at 10 mM.Different concentrations of Co2+ and K+ can significantly inhibit the enzyme activity,and the maximum enzyme activity loss is about 50%-60%.Low concentrations of Mn2+,Cu2+and Ca2+ have little effect on the enzyme activity of the recombinant enzyme,but high concentrations of Mn2+,Cu2+ and Ca2+ have significant inhibitory effects on enzyme activity.