Construction And Application Research Of Engineered Bacteria Displaying Bifunctional Aminopeptidase Aps On The Surface Of Bacillus Subtilis Spores

Microbial surface display technology is a technique that fuses the target protein gene with cell surface proteins genes on microbial cells through gene cloning to express and display the fusion protein on the surface of microbial cells.Displaying protease on the surface of Bacillus subtilis spores not only avoids the complicated and expensive process of enzyme separation and purification,but also gives the protease more stability due to the stress resistance of the spores.The bifunctional aminopeptidase Aps is a thermotolerant aminopeptidase screened from Pseudomonas aeruginosa GF31 in the early laboratory stage,which can degrade pyrethroids pesticide.It exhibits enzymatic hybridization,acting as both an aminopeptidase that hydrolyzes peptide substrates represented by Leu-p NA,with wide application prospects in multiple fields such as food,medicine,and the environment,and an esterase that can degrade various kinds of pyrethroids pesticide and has potential applications in environmental bioremediation.This study utilized surface display technology to display the enzyme on the surface of Bacillus subtilis spores with the aim of obtaining a new type of whole-cell biocatalyst that is easy to separate and highly stable,laying a foundation for practical applications of this enzyme.The research content of this article involves the following three aspects:(1)Construction of spore surface displaying aminopeptidase engineering bacteria: By optimizing the host strain,anchor protein,and plasmid vector,five active recombinant spores were successfully constructed.Among them,Bacillus subtilis WB600-PMA5-cot G-Aps was selected as the strain with the highest aminopeptidase activity.Immunofluorescence microscopy and Western Blot were used to locate the target protein,demonstrating its correct localization on the spore surface.Subsequently,the expression conditions of the engineered strain were optimized,and it was found that the optimal aminopeptidase activity expression was achieved by culturing at 37℃ and 220 r/min for 72 h.(2)Peptidase activity assay and application research of surfacedisplayed aminopeptidase: The peptidase activity of the surface-displayed aminopeptidase was measured under optimal reaction conditions of 60℃and p H= 9.0,with a maximum activity of 229.97 U/g spore and a half-life of approximately 22 h.The enzyme exhibited good storage stability at 4℃,room temperature,and-20℃,with the best stability observed at 4℃,where relative enzymatic activity remained at 64.35% after 18 weeks of storage.In addition,the surface-displayed aminopeptidase could tolerate 3 mol/L Na Cl solution,which was beneficial to expand its application range.The aminopeptidase and alkaline protease were used in a synergistic hydrolysis of soybean protein,in which 5.0% of soybean protein was hydrolyzed for8 h at 60℃ with an enzyme ratio of 2:1 between the alkaline protease and the aminopeptidase,with an initial hydrolysis p H of 10.0 followed by 9.0.The maximum hydrolysis degree was 55.50%,which was 3.3-fold and 1.5-fold higher than that achieved by surface-displayed Aminopeptidase or alkaline protease alone,respectively.Simultaneously,the content of hydrophobic amino acid,including Leu,Tyr,and Phe,increased by 22.12mg/L,9.47 mg/L,and 19.86 mg/L,respectively,while umami amino acids,including Glu and Asp,increased by 15.06 mg/L and 6.33 mg/L,respectively.These results suggested that surface-displayed aminopeptidase significantly contributes to the deep hydrolysis of proteins and enhances their taste by reducing bitterness and increasing freshness in the hydrolysate.(3)Determination and application of the degradation activity of surface displayed aminopeptidase on pyrethroid insecticides: The surfacedisplayed aminopeptidase exhibited the ability to degrade various pyrethroid insecticides such as cypermethrin,deltamethrin and fenpropathrin,with the best degradation effect observed for β-cypermethrin,under optimal conditions of 60℃ and p H= 8.0.The degradation of β-cypermethrin in water,soil,and on the surface of fruits and vegetables was investigated,with the following results: under optimal conditions,the degradation rate of 50 mg/L β-cypermethrin in water was56.5% after 72 h;the highest degradation rate of 76.28% was achieved for50 mg/kg β-cypermethrin in sterilized soil after 10 days;the degradation rates of low residue levels of β-cypermethrin on the surfaces of fruits(cucumber)and vegetables(lettuce)were 57.49% and 47.02%,respectively,while the degradation rates of high residue levels were 62.79%and 52.14%,respectively.These results suggested that the surfacedisplayed aminopeptidase has potential applications in environmental bioremediation and emergency treatment of food pesticide residues.