Enzymatic Production Of 5'-Inosinic Acid By A Recombinant Acid Phosphatase/Phosphotransferase

Nonspecific acid phosphatases(NSAPs)is a widely used acid phosphatase,which can catalyze hydrolysis,transphosphorylation reactions.It has great specificity and broad substrate spectrum.It is popular both in theoretical research and industrial application.NSAPs opens up more applications in various fields,including the synthesis of 5'-inosinic acid(5'-IMP).5'-IMP was often used as food additives and pharmaceutical intermediates.Currently,screening and exploration of AP/PTase and its industrial applications for the preparation of 5'-nucleotides in biotechnology have been hot issues and another key area of interest in nucleotides production field.In this paper,we synthesized an acid phosphatase from Escherichia blattae JCM 1650 and optimized its codons,the total length of nucleotide sequence was 747 bp,encoding 248 amino acids.Through the recation of digestion analysis,the recombinant E.coli BL21(DE3)/pET28b-AP/PT was successfully constructed.Recombinant expression was induced by IPTG and analyzed by SDS-PAGE.The result indicated the molecular mass of the recombinant enzyme was about 25 kDa,and specific activity was 500 U/g.To improve the enzyme production of E.coli BL21(DE3)/pET28b-AP/PT,single factor experiment was used to optimize the fermentation process for the production of acid phosphotransferase from E.coli BL21(DE3)/pET28b(+)-AP/PT.The results showed that the optimal fermentation and expression parameters were as follows:glycerol 10.0 g/L,peptone 15.0 g/L,yeast 10.0 g/L,NaCl 10 g/L,initial pH 7.0,IPTG concentration of 0.1 mM,induction temperature of 28? for 10 h.The resulting acid phosphotransferase activity could reach up to 145.6 U/L and was 2.1 times higher than that before the optimization.After expressed,the recombinant AP/PTase was purified using Nickel-NTA.The characteristics investigation of purified AP/PTase showed that the optimal temperature and pH of this enzyme were 30?and 5.0,respectively.The recombinant AP/PTase was stable at pH 5.8 and the half-life of the purified enzyme at 30,40,50,and 60? were 3.2 h?0.9 h?0.23 h?0.076 h,respectively.The activity was partially inhibited by heavy metal ions such as Hg2+,Ag+ and Cu2+,but not by chelating reagents such as EDTA.In addition,the results showed that addition of Tween-20,Tween-80 and Triton X-100 could slightly activate the activity of acid phosphotransferase.However,DTT could inhibite the activity of acid phosphotransferase,which showed thiol groups may play an important role in the function of the active site because DTT is sulfhydryl reagents.The values of Km and Vmax for inosine were 40 mM and 3.5 U/mg,respectively.Moreover,the phosphorylation of inosine by resting cells of E.coli BL21(DE3)/pET28b(+)-AP/PT were optimized.The result showed that the optimal temperature and pH were 35? and 4.0,respectively.In addition,the optimal concentration of inosine and disodium hydrogen pyrophosphate were 10 mg/mL and 200 mg/mL,respectively.Based on these conditions,the molar yield of 5'-IMP reached 83.3%.This study laid a good foundation for enzymatic production of 5'-IMP.After isolation and purification of 5'-IMP,the structure of 5'-IMP was confirmed using IR,NMR.