The Study Of One Step Purification-immobilization And Molecular Modification Of Lysine Decarboxylase

1,5-diaminopentane(DAP)is an essential precursor for production of quinazine,chelating agents and additives,etc.The most significant application is to produce polyamides(nylon).Polyamides have a huge market all over the world,and 6.6 million tons of them are consumed every year.The bio-based nylon PA56 synthesized from DAP has superior properties and it is a perfect substitute for traditional nylon.The application of DAP may reduce the environmental pollution and the consumption of nonrenewable resources.Biological method plays an important role in production of DAP,and the lysine decarboxylase(LDC)is the core enzyme in the procedure.However,the low stability and easy inactivation of the lysine decarboxylase limit the high yield of DAP.To overcome this problem,in this study,firstly,the lysine decarboxylase fused with 6*Histidine tags was overexpressed in Escherichia coli,and immobilized within the stable barium-alginate beads which severed as the matrix for immobilized metal ion affinity chromatography(IMAC)without chelating agents.After one step purification and immobilization,the activity of immobilized and free lysine decarboxylase was determined,and the kinetic parameters were calculated.The purification process for the lysine decarboxylase was highly specific,and the p H stability was enhanced.The immobilized lysine decarboxylase could achieve >60% of its initial activity even at p H 8,while the free lysine decarboxylase remained only 40% of its initial activity at the same condition.Besides,the affinity of immobilized enzyme to substrate was higher than the free one.Afterwards,the lysine decarboxylase was modified by EKylation.The C terminal of the lysine decarboxylase was connected with an alternating peptide of lysine and glutamate(EK)which was the analogue of the zwitterionic polymers.The EK peptide could provide a hydrophilic microenvironment for lysine decarboxylase and absorb the water from the hydrophobic core,thus enhanced the hydrophobic interaction and structural rigidity.As a result,the enzyme stability increased substantially.The absolute activity of modified lysine decarboxylase was doubled.The activity of modified lysine decarboxylase in extreme conditions(such as p H 8 and 65?)was higher than the activity of the native one in optimum conditions.In addition,the tolerance to acid,alkali,organic solvents and denaturant was also improved.After incubation in those reagents,the remaining activity of modified enzyme was 2-7.4 times higher than that of unmodified one.After being preserved at 4? for 21 days,the modified lysine decarboxylase remained 50% of its initial activity,but the wild type enzyme lost its activity totally.Beyond that,the modification could reduce the inhibitory effect of Llysine on the lysine decarboxylase,yielding more DAP at high substrate concentration.Finally,the online protein analysis tools were used to predict the physicochemical properties for modified lysine decarboxylase,and the molecular simulation using the software Amber18 was performed to predict the docking model,RMSD,binding energy,catalytic distance.A “HugHandshake” model was proposed to demonstrate the change of kinetic parameters and the improvement of stability.The modification of lysine decarboxylase by connecting the EK peptide increased the catalytic activity and stability,laying a good foundation for engineering synthesis pathway of DAP and constructing the strain with a high DAP yield.