Biocatalysis Of 5'-inosinic Acid By Recombinant Escherichia. Coli Expressing Phosphatase/phosphotransferase

Nonspecific acid phosphatases(NSAPs)is a widely used acid phosphatase,which can catalyze hydrolysis and transphosphorylation reactions.It has high specificity and broad substrate spectrum.It is popular both in theoretical research and industrial application.NSAPs could be applied in various synthesis fields,such as synthesis of 5'-inosinic acid(5'-IMP).5'-IMP was widely used as food additives and pharmaceutical intermediates.Currently,interests in large scale production of 5'-nucleotides with phosphatase/phosphotransferase(AP/Ptase)aroused.In this paper,recombinant E.coli BL21(DE3)/pET28b-AP/PT which can express acid phosphatases was used.Firstly,The cultivation conditions of E.coli BL21(DE3)/pET-28b-AP/PT for enzyme production were optimized in shake flask.The optimal cultivation medium was as following(g/L):glycerol 7.5,peptone 12.5,yeast 10.0,NaCl 10,ZrnCl20.1,initial pH 7.0.The optimal induction conditions were IPTG 0.1 mM,28? and 10 h.Under the cultivation conditions,the biomass and enzyme activity 3.31 g DCW/L adn 143.8 U/L,which were almost 1.4 times and 2.0 times of those obtained under initial fermentation conditions,respectively.On the base of above results,batch fermentation processes were carried out in 5 L fermenter.The optimal fermentation conditions were as follows:seed inoculation size 3%(V/V),cultivation temperature 37 ?,inducer lactose 10 g/L,induction temperature 28 ?,induction time OD600 7,500 r/min,aeration 1.5 L/min,seed age 8 h.Under above conditions,the final biomass and enzyme activity reached 7.55 g DCW/L and 232.54 U/L,respectively.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.The results showed that the optimal fermentation and expression parameters were as follows:glycerol 7.5 g/L,peptone 12.5 g/L,yeast 10.0 g/L,NaCl 10 g/L,ZnCl2 0.1 g/L,initial pH 7.0,IPTG concentration of 0.1 mM,induction temperature of 28? for 10 h.Under the optimized medium and culture conditions,cell dry weight reached 3.31 g DCW/L,Enzyme activity reached 143.8 U/L,which is almost 1.4 times and 2.0 times of those under initial fermentation conditions,respectively.In addition,we study the batch fermentation process in 5L fermenter.Stirring speed:500 r/min,ventilation volume:1.5 L/min,seed age:8 h,inoculation amount:3%,induction timing:about OD600=7,the concentration of the inducer:lactose 10 g/L,and finally the biomass reached 7.55 g DCW/L,Enzyme activity reached 232.54 U/L.Finally,the recombinant cells were further immobilized to improve their operation stability.The optimal immobilization method was polyvinyl alcohol(PVA)-sodium alginate(SA)-active carbon(AC)entrapment.The optimal immobilization conditions were:2.0%sodium alginate,6.0%polyvinyl alcohol,2%CaCl2,6.0%wet cell.The optimal reaction conditions were 1.0%substrate,35?,pH 4.0.The immobilized cells possessed excellent operation stability.After 12 cycles,about 54.5%of activity was remained,whereas free cells completely lost all activity after 7 cycles.