Cell Surface Display Of Carbonic Anhydrase And Its Enzymology Properties

Capturing CO₂ by carbonic anhydrase（CA）is one of the most promising technology at present.However,pure enzymes have high purification cost,poor stability,easy to be inactivated and cannot be recycled,the intracellular enzymes have shortcomings such as outer membrane barrier and low catalytic efficiency,which limits the application of pure enzymes and intracellular enzymatic methods.To address these questions,this study intends to display CA on the cell surface by genetic engineering methods to construct a new type of whole cell biocatalyst.Firstly,ice nucleoprotein（INPN）was used as a carrier protein to construct a CA surface display strain.We designed and constructed 18 CA-expressing engineering bacteria from the aspects of host strain,protein fusion method,CA source,signal peptide,solubilizing protein and et al.The engineering strains were screening originally by measuring whole cell activity,and then the optimal system for CA surface display was obtained by immunofluorescence microscope and cell membrane integrity analysis:E.coli BL21 was used as the host strain,and the INPN and the target protein were fused through the intermediate repeat sequence and the Flexible Linker.The p ET22b plasmid with the signal peptide Pel B was used for intracellular expression and cell expression of Sulfurihydrogenibium azorenseα-type carbonic anhydrase（SazCA）.Through the cell fractionation and Westen-Bloting analysis,it was determined that SazCA and INPN were successfully anchored on the outer membrane of the cell as a fusion protein.For the selected carbonic anhydrase surface display strain E-22b-I_RLS and intracellular expression strain E-22b-S,conditions were optimized for the four aspects of the IPTG concentration,induction temperature,Zn SO4 concentration and induction time.The optimal induction temperature of E-22b-I_RLS and E-22b-S is 25℃,the optimal IPTG concentration is 0.4 m M,the optimal Zn²⁺concentration is 0.5 m M,and the optimal induction time is 24 h and 12 h,respectively.Under the optimal induction conditions,the enzyme activity of the surface display strain E-22b-I_RLS(9.402Um L^-1OD^-1₆₀₀)was significantly higher than the intracellular expression strain E-22b-S(6.073 Um L^-1OD^-1₆₀₀).Stability analysis shows that SazCA surface display engineering strain E-22b-I_RLS has higher thermal stability and higher p H stability compared with free SazCA.The long-term stability of the enzyme after surface display was also significantly improved.In order to prove the CO₂ capture and mineralization ability of the engineered strain displayed on the surface,the engineered strain and free enzymes were used in the CO₂ mineralization experiment.The amount of CaCO₃ produced by different mineralization systems at 25℃ for 10 minutes was:free SazCA>E-22b-I_RLS>E-22b-S.The amount of CaCO₃ deposition（241 mg）catalyzed by the strain E-22b-I_RLS on the surface was significantly higher than the intracellular expression strain E-22b-S（173 mg）.In conclusion,we successfully displayed CA on the surface of E.coli and constructed a new whole cell biocatalyst.The enzyme activity of the whole-cell catalyst is increased compared with intracellular enzymes,and increasing the rate of catalyzed CO₂ mineralization further;compared with free SazCA,the stability of the enzyme is improved.CA surface display engineering strain can be directly applied to industrial processes,and the preparation and purification process of enzymes can be removed through centrifugation recovery,and the application cost can also be reduced.