Biosynthesis Of Iminodiacetic Acid From Iminodiacetonitrile By Recombinant Escherichia Coli Harboring Nitrilase

Iminodiacetic acid as an important intermediates of the chelating agent,is widely used in the production of glyphosate herbicide and surfactant.Nowadays,the current production capacity of iminodiacetic acid is far from being able to meet demands,as the sales of glyphosate increased by year.Traditionally,the chemical manufacturing process of IDA employs a strong base catalyst,resulting in the by-product of salts due to the neutralization of the base used in the final product separation,which causes serious environmental problems.Compared to chemical synthesis of IDA,nitrile-converting biocatalysts have attracted more attention because of its high activity,mild reaction conditions and environmental compatibility.Today,the world's development theme is energy conservation and environmental protection,so biological hydrolysis of nitrile have attracted unprecedented attention.This paper is aim to constract a recombinant E.coli harboring nitrilase,which can biosynthesize iminodiacetic acid.Firstly,the nitrilase genes from Acidovorax facilis are extracted by genetic engineering technology.Then the gene of this nitrilase with the length of 1121bp,which encodes 374 amino acids,has been successfully cloned.After constracting the nitrilase gene into expression vector pET-28b(+),pET-28b(+)-NIT had been successfully transferred into E.coli BL21(DE3).SDS-PAGE analysis shows the size of nitrilase is approximately 42kDa.The enzyme activity of recombinant E.coli is 72 U/g(DCW),and the enzyme activity of purified nitrilase is 89 U/mg protein.We optimized the culture conditions of E.coli BL21(DE3)/pET-28b(+)-NIT for enzyme production.The optimized results are(g/L):peptone 15,yeast extract 10,(NH4)2SO4 5,NaCl 10,glycerol 10,KH2PO4 1.5,K2HPO4.3H2O 2.3,MgSO4.7H2O 0.375,initial pH 7.0-7.2,the liquid volume of shake flasks is 50mL/250mL.The optimized induced conditions are 0.1mM IPTG,28?.Under the optimized medium and culture conditions,cell dry weight reached 5.48 g/L(DCW),activity reached 86.66 U/g(DCW)after 20h induction,which is almost 2.4 times and 1.2 times of those under initial fermentation conditions,respectively.In addition,we study the catalytic properties of recombinant E.coli.It is showed that the optimum catalytic temperature is 45?,when the temperature reaches 70?,the enzyme is almost completely lost activity,the enzyme shows the best thermal stability when the temperature is 35?,Tm value is 57?.The optimum range of pH is 6.5-7.5,the enzyme stays stable at pH 6.5.The nitrilase activity could be completely inhibited by Hg2+ and Ag+.Finally,the immobilization of E.coli BL21(DE3)/pET-28b(+)-NIT had been studied to improve the stability of operation.The most appropriate method of immobilization is polyvinyl alcohol(PVA)-sodium alginate(SA)entraption,which showed similar catalytic properties to free cells(residual activity of 98.1%).The maximum relative nitrilase activity was futher observed at 1.0%sodium alginate,8.0%polyvinyl alcohol,10%CaCl2,5.0%wet cell,and 1.0%substrate concentration at temperature of 40?,while the free cells showed the optimum temperature at 45 ?.It is also showed the optimal pH is 6.5,which shifted to acidic acid,the optimal pH of free cells is 7.0.Both free and immobilized cells could not endure strong acid and alkali conditions,and both relative activities were lower than 6%when immobilized and free cells immersed pH 3.0 or pH 10.0 for 1h.The results showed that the operational efficiency of immobilized cells remained stable and approximately 35%of activity was remained after 10 reuse cycles,whereas free cell completely lost all activity when the reuse cycle was 9.At last,we expanded immobilized cells to airlift bioreactor,the optimal catalysis time is shifted to 10 hours.When the concentration of iminodiacetonitrile is 1%,the residual activity is 60%after 5 batches.