Using Self-assembled Bi-enzyme Complex With Tag-Catcher To Prepare D-amino Acid Efficiently

Amino acids can be divided into D-amino acids and L-amino acids according to their chirality.D-amino acids were considered unnatural and useless initially.However,with the development of technology,the application of D-amino acids is becoming more and more widely used.The D-amino acid bioconversion has become a hotspot in research.In our laboratory,an engineered strain which can express two enzymes,D-hydantoinase(HYD)and D-carbamoylase(CAB),has been constructed,it is used to prepare D-amino acid.Nevertheless,the activity and stability of CAB limit its industrial application.To find an optimized CAB has become an urgent need at present.In this study,CAB(ATO)gene sequence was designed and synthesized with directional combination mutation,which was optimized for CAB in antioxidant sites(M5L,M31 L,M239L,M244L),solubilization sites(A18E,Y30 D,K34E)and heat resistant sites(Q12L,Q23 L,Q207E,H58 Y,P204E,V237 A,N242G,H248 Q,T262A,T263 S,E266D,T271 I,A273P).SDS-PAGE analysis showed CAB(ATO)had higher soluble expression level in E.coli BL21(DE3)than CAB by 17%.The activity of p QE30-cab(ATO)/E.coli BL21(DE3)was 127% higher than that of p QE30-cab/E.coli BL21(DE3).At the same time,the oxidative resistance and thermostability of CAB(ATO)was significantly improved compared with the wild type enzyme CAB.The peptides,SpyTag and SpyCatcher derived from S.pyogenes fibronectin have been proved that they can spontaneously form isopeptide bonds.This property of them can be used to prepare self-assembled multi-enzyme complexes.To figure out whether SpyTag/SpyCatcher would affect enzyme activity,we fused SpyCatcher to the C-terminal of HYD and SpyTag/SnoopTag to the N-terminal of it to obtain SPT-SL-HYD-LL-SPC and SNT-SL-HYD-LL-SPC respectively.The results of SDS-PAGE showed that the apparent molecular weight of SPT-SL-Hy-LL-SPC is 66 kDa,which is slightly smaller than that of uncycled protein SNT-SL-HYD-LL-SPC(71k Da).That indicated the isopeptide bond between Spytag and Spycatcher in SPT-SL-HYDLL-SPC could be spontaneously formed in cell,and stable cyclized proteins were obtained.The substrate specificities of them were changed and the enzyme activity of SPT-SL-HYD-LL-SPC and SNT-SL-HYD-LL-SPC was reduced by 77% and 71% respectively.In the consideration of the activity of HYD was much higher than CAB in the engineered strain,the decrease in the enzyme activity of HYD had no effect on the conversion efficiency of the engineered strain.On the basis of the above experiments,we constructed and expressed the recombinant enzymes SPT-SL-HYD and SNP-SL-HYD by fusing His-SpyTag and His-SnoopTag at the N terminal of HYD.And CAB(ATO)-LL-SPC was obtained by fusing SpyCatcher-His at the C terminal of CAB(ATO).The results showed that the activity of self-assembled bi-enzyme complex was 35% higher than no-assembled bi-enzyme mixture,and was 1.62 times higher than the original engineered strain.SPT-SL-HYD,SNT-SL-HYD and CAB(ATO)-LL-SPC were purified through Ni-chelating affinity chromatography.When CAB(ATO)-LL-SPC was mixed with SPT-SL-HYD or SNT-SL-HYD at 42 ? in vitro,it had the highest self-assembled efficiency.The results showed that the enzyme activity of the bi-enzyme complex with more stability was 31% higher than that of the no-assembled bi-enzyme mixture.