Refolding And Immobilization Of An Bifunctional Acid Urease Expressed In E.coli BL21 (DE3)

Bifunctional acid urease can hydrolyse urea and EC,the latter is a potential carcinogen.In this study,acid urease was successfully expressed in E.coli BL21?DE3?,but most of them were in the form of inclusion bodies with few soluble proteins.The inclusion bodies was washed and dissolved for refolding,and recombinant acid urease was successfully refolded.The obtained acid urease was immobilized on graphene oxide?GO?/chitosan?CS?composite beads,and preliminary application of immobilized acid urease was made in fluidized bed reactor to removel urea and EC of rice wine.First,pET-42a-ureABCEFGD was successfully constucted and expressed in E.coli BL21?DE3?,but most of them were in the form of inclusion bodies with few soluble proteins.The higher purity protein was obtained after s unconsolidated inclusions bodiescontain more impurities was cleanined for two times.The recovery of protein reach 95%when the purity proteins were dissolved in buffer containing 8 mol urea,0.05 mmol·L^-1 DTT,0.05 mol Arg.Three methods were studied to refold acid urease in vitro based on traditional dilution refolding.In dilution refolding,it is found that it got hightest activity in refolding solution containing 50 mmol·L^-1 Tris–HCl?pH8.5?,1.5 g·L^-11 Arginine,0.5 mol urea,1 mol NaCl,0.4mmol·L^-1 GSSG and 4 mmol·L^-1 GSH,20%?v/v?glycol.In dialysis refolding,the highest activity was obtained when when refolding in 35 times buffer for 48 h.In dilution-ultrafiltration refolding,the highest activity was obtained when centrifuge at 2000rpm·min^-1 based on dilution buffer.Compared with dilution refolding,dialysis refolding and dilution-ultrafiltration refolding,the specific activity of urease and ureathanase were 8.1±0.8?2.7±0.3 U·mg^-1;10.8±1?3.4±0.3 U·mg^-1;12.3±1?3.8±0.5 U·mg^-1,respectively after purified with Ni-NTA.The total yield of protein were 24±5%?22±4%?21±4%,repectively.The refolded protein closely regained their native structure which was ascertained through CD spectrum analysis.The obtained acid urease were immobilized on graphene oxide/chitosan?GO/CS?composite beads,the mixing ratio of the two substances is studied.When the ratio of weight was 3/2,the highest enzyme activity was obtained after fixed time was 12 h and glutaraldehyde concentration of 2.5%,GO and CS reaction time was 8h,indicating that under these conditions GO-CS can be of well crosslinking reaction with the enzyme.The temperature of immobilization slightly influenced the recovery rate of activity.The reaction under 4?obtains the higher recovery than under 25?,but it takes longer time.The gentle reaction can make urease react uniformly with GO-CS composite.From steady-state kinetics,with time-dependent thermal inactivation studies,the t_1/2?Half-life?of immobilized acid urease at 65?was 400 min and the t_1/2?Half-life?of decay in urease activity was 12 h,43 day for soluble,GO/CS immobilized urease.The immobilized acid urease activity reserved 80%in buffer containing 20%ethyl alcohol.Meanwhile,only 10%of the original activity of graphene oxide/chitosan–urease beads lost after the beads was used for 10 times,indicating excellent reusability.A significant change in Km occured during the immobilization,which were 9.14 mmol·L^-1 for urea,386 mmol·L^-1 for EC in free states,and5.28 mmol·L^-1 for urea,180 mmol·L^-1 for EC in immobilized state,respectively.The values of V_max for immobilized and soluble ureases were 1.08?mol·min^-1 to EC,1.16?mol·min^-1protein to urea and 0.782?mol·min^-1 to EC,0.931?mol·min^-1 to urea,respectively.To inspect result of immobilized urease in fluidized bed reactor for removing urea and EC in rice wine.When 50 ml rice wine were processed in column,the urea and EC removal rate reach 93.85%and 57.46%when 20 U immobilized acid urease was added in the flow rate of 0.2 m L·min^-1 after four times of column operation were made.Well performances make it possible for further industrial applications.