Molecular Modification To Acid Phosphatase And Its Application In The Production Of 5'-IMP

Flavor nucleotides is one of the food additives,in addition to improve the freshness of food,it can also improve the taste,and make the taste more close to the natural foods.Among them,the 5'-IMP has been widely used in food additives and pharmaceutical intermediates.Biological catalysis on acid phosphatase to produce flavor nucleotides has a lot of advantages such as mild reaction conditions,low cost,low toxicity and high specificity,and it also conforms to the development direction of green chemistry,which is friendly to environment.Therefore,it has become the focus of attention.With inosine and PPi as substrates,it is a new way to acid phosphatase catalyzes the production of 5'-IMP.In this paper,we used the recombinant E.coli BL21(DE3)/pET28b-AP/PT as the original strain.At first,we searched the protein crystal structure of acid phosphatase in the PDB library,and then with the model protein(PDB ID:1EOI)and in inosine molecule we did molecular docking by using the program Discovery Studio 2.5.From the results based on improving the enzyme activity,we chose the feasible mutational sites and designed the primers,then used the site-saturation mutagenesis to achieve molecular modification.Finally,we got the mutant strain E.coli BL21(DE3)/pET28b-AP/PT-D108S/N143L.Because the recombinant protein was His-tagged,we used Ni-NTA column for purifing acid phosphatase.With the purified enzyme,we investigated its enzymatic properties,including the optimum temperature,optimum pH,pH stability,thermal stability,reaction kinetic parameters,the influence of metal ions on the enzyme activity,etc..Final results showed that the optimum temperature of the enzyme was 30?,.The optimum pH was 4.0,and the enzyme activity and stability under the condition of alkaline were low.When adding metal ions and surfactant into the reaction system,Ni2+ had the promoting effect to enzyme activity,while Cu2+and EDTA had a slight inhibition on enzyme activity.The values of Km and Vmax for inosine were 5.2 mM and 30.12 U/mg,respectively.Through the single factor experiment of mutant strain E.coli BL21(DE3)/pET28b-AP/PT-D108S/N143L to optimize of enzyme production conditions,we found the optimum medium composition(g/L):peptone,14,yeast,5,NaCl,10,glycerin 5,and the initial pH 7.0 of the medium.And the best induced condition:IPTG concentration of 0.8 mM,induction temperature of 28? for 12 h.Finally the enzyme activity of the mutant strain was 1832.82 U/L which was 1.2 times higher than before the optimization and the biomass(DCW)was 4.85 g/L that was 2.3 times higher than before.Batch fermentation of 5 L fermenter fermented with the conditions was 2%inoculum size,1.5 L/min VVM,500 rpm,and the induction time was about OD600=6.Finally mutant strain of enzyme activity and biomass were 5080.02 U/L,14.04 g/L,it was basically the same as shaking flask fermentation.At last we researched the catalytic conditions to produce 5'-IMP.The results showed that the optimum temperature and pH were 35? and 4.0,respectively.In addition,the optimal concentration of inosine and sodium pyrophosphate were 35 mg/mL and 250 mg/mL with 60 g/L the concentration of bacteria.Based on these conditions,the yield of 5'-IMP reached 90.6%.