Catalytic Performance Of Covalent Oligomeric Enzyme And The Renaturation Of Cyclized Enzyme

Oligomeric Foldon assisted enzymes oligomerization could improve the catalytic performance and the purification yield when combined with elastin-like polypeptides(ELP),which is a simple and feasible enzyme engineering method.However,the non-covalent bond between the Foldon monomers usually ruptured once in high temperature and caused the low catalytic improvement.Therefore,we mutated the amino acid at proposition position into cysteine to induce disulfide bonds and enhance its thermostability;the application of Foldon in thermophilic enzymes could be developed by this way.Besides,in order to explore the novel methods in protein renaturation,we assay the renaturation efficiency of cyclized enzyme under different denaturation conditions.The result as follows:(1)By analyzing the structure of Foldon,we constructed a more stable oligomeric ELP-Foldon(E-Fm)through substituting the Arg8 and Gly18 of Foldon into cysteines,and verified that the disulfide bonds informed between the Foldon monomers by experiment.Then we constructed linear lichenase(B-E),non-covalent oligomeric lichenase(B-E-F)and covalent oligomeric lichenase(B-E-Fm)and purified them by ITC.The optimum temperature and pH were 45? and 7.0,respectively.The B-E-F depolymerized at 40? while the B-E-Fm at 70?.Besides,the result of kinetics showed that oligomeric enzymes performed a better catalytic efficient and the B-E-Fm best below 55?.Once the lichenase was inactive at 60?,the catalytic efficient of three enzymes was decreased obviously.(2)To investigate the performances of the modified Fodon at high temperature,we selected a thermophilic xylanase(X11A)with a higher optimum temperature,and constructed the linear xylanase(X11A-E),non-covalent oligomeric xylanase(X11A-E-F)and covalent oligomeric xylanase(X11A-E-Fm)then purified them by ITC.All of the optimum pH were 7.0,and the optimum temperatures of X11A-E,X11A-E and X11A-E-Fm were 60?,55? and 55?,respectively.The catalytic efficient of X11A-E-F was the same as X11A-E at 60? but X11A-E-Fm was better than the other two at 60-65?.The result proved that Fm could improve the catalytic efficiency of the thermophilic enzyme.(3)In addition,under the different denaturation conditions,the loss of the activity of the linear lichenase(B-E)and cyclized lichenase(cB-E)increased with the increase of solvent polarity.Methanol,benzyl alcohol,acetone and 1 mol/L HCl could make B-E and cB-E inactivate in a short time.1-butanol and ethylene glycol have different degrees of influence on enzyme activity,respectively.Isopropyl alcohol had little impact on the activity of cB-E,which would remain more than 80% after incubated for 1 h.Then,we removed the denaturant,B-E and cB-E scarcely reactivate after denatured by methanol.However,they almost reactivate totally after incubated by ethanol,acetonitrile,isopropyl alcohol and 1-butanol.It was interesting that the cB-E could recover over 80% of the activity after denatured by Benzyl alcohol,acetone and 1 mol/L HCl in 10 min,but B-E not.The results confirmed that the catalytic efficient of thermophilic enzyme was improved through covalent bonds oligomerization,and has great potential in thermophilic enzyme engineering.Besides,the results of renaturation indicated that the cyclized enzyme could renatured rapidly.It's expected that we could establish a new and commonly protein renaturation method using the cyclized protein design,and may provide a new idea for the field of protein renaturation.