Expression And Characterization Of A Lysine Aminopeptidase In Escherichia Coli

Lysine aminopeptidase?LAP?is a type of exo-protease which can hydrolyze peptide bond from the N-terminus of polypeptide,and is particularly efficient in cleavage of the lysine residue to release free lysine.LAP can be used as a flavourzyme to remove the bitter peptide of protein hydrolysate,and thus increase the products' taste.It can be also applied to fodder processing,medical diagnosis,protein sequencing,skincare,etc.In short,LAP hold high value both in studies and applications.Thermo-stable enzyme can retain relatively activity in high temperature condition,so it exists broad prospects in industrial manufacture for cost reduction,technique simplification and productivity improving.In this study,the lap gene was cloned by two specific primers using Pseudomonas aeruginosa NJ-814 genome as template.Recombinant plasmid pET42a-lap was transformed to prokaryotic expression system E.coli BL21?DE3?and recombinant lysine aminopeptidase was successfully expressed.After inducing under 16°C,220 r·min-1 for 72 h,the recombinant strain BLAP cultured in fermentation medium?within 50 ?g/mL kanamycin?can obtain 2.54 U·m L-1 intracellular enzyme activity.Through extracellular secretion strategies,such as molecular chaperone construction and surfactant addition,we discovered that adding 1% sorbitol is helpful to promoting secretion of the recombinant enzyme.The recombinant aminopeptidase was purified 4.7-fold with a recovery of 83.5% after separation by Ni²⁺-NAT affinity chromatography.The enzymatic characterization research showed the purified enzyme exhibits the highest catalytic activity at pH 8.5 and 80°C,it remains stable between pH 6.0¹0.0 under 70°C.Ni²⁺,Zn²⁺,reductant DTT,?-mercaptoethanol and metal chelating agent EDTA showed strong inbition on the activity of the purified aminopeptidase.Mn²⁺,Ca²⁺,Cu²⁺ and Serine proteinase inhibitor PMSF hold limited effects on the recombinant enzyme,while Mg²⁺ and Co²⁺ can activate the enzyme activity,Co²⁺ can especially raise the activity to 4 times as high as the original result.This recombinant enzyme can hydrolysis 3 types of substrate?Arg-pNA,Leu-pNA,Lys-pNA?and Lys-pNA is the optimal substrate.Enzyme kinetic analysis shows the Km and Vmax of LAP with Lys-pNA as substrate were 4.86 mmol·L-1 and 8.76 ?mol·L-1·min-1 respectively.The optimal medium for expressing recombinant lysine aminopeptidase at shake flask stage is listed as below: 0.5% glucose,2% peptone,3% yeast extract,55 mmol·L-1 K2HPO4,17 mmol·L-1 KH2PO4,0.2 mmol·L-1 CoCl2,initial pH 7.5,culture volume 40 m L/250 m L.Add 0.5 mmol·L-1 inducer IPTG to the fermentation liquor after 8 h cultivation at 37°C,220 r·min-1 with 2% inoculum size,then transfer the fermentation liquor to 16°C,240 r·min-1 incubator shaker inducing the recombinant enzyme expression.After optimizing,the recombinant enzyme activity reached 3.47 U·m L-1,1.37 times than before.An determinate mutation strategy was designed by whole plasmid PCR for removing the Protease Associated domain,and then we obtained an mutational lysine aminopeptidase with better thermo-stable ability.In the meantime,we studied the application of rice protein hydrolyzing by the mutational lysine aminopeptidase and alkaline protease,the result shows that high molecular peptidase was degraded and free amino acid in the hydrolysate was significantly increased.