Study On Separation,Purification,Dynamic Structure And Antibacterial Activity Of ?-poly-L-lysine

?-poly-L-lysine(?-PL)consists of 25-35 lysine residues which are linked by isopeptide bonds formed by dehydration condensation of ?-carboxyl and ?-amino groups,having good antibacterial activity and broad-spectrum inhibition range.As a natural and safe biological preservative,it has become a research hotspot in recent years,and its output has been significantly increased after years of strain breeding and fermentation strategies optimization.However,insufficient research on subsequent separation and purification processes has resulted in lower purity and recovery of?-PL;at the same time,the structure and antibacterial mechanism of ?-PL,which is one of the only two homologous amino acid polymers in nature,have not been revealed clearly.In this study,?-PL was taken as the object of the study to optimize its industrial green short-process production,study the dynamic secondary structure and antibacterial activity,which laid a foundation for reducing production cost and product application development of ?-PL.The main research results obtained in this paper are as follows:Research on separation and purification.The whole production is divided into three stages:solid-liquid separation-ion exchange-desalination drying.The 10 KDa polysulfone ultrafiltration membrane was used in the solid-liquid separation stage,which separated impurities to the greatest extent and reduced the contamination of subsequent ion adsorption resins,resulting in a decrease in exchange capacity.In the ion exchange stage,the secondary anion column was removed,and 40%neutral ethanol was added to elute and remove impurities.The purity of the product reached 98%,and the amount of acid,alkali and water was decreased.IRC50 adsorption capacity reached 0.17 g ?-PL/g resin(dry weight).3000 Da polysulfone ultrafiltration membrane was used in the decolorization and desalination stage,which solved the problems of low recovery rate of activated carbon and macroporous adsorption resin and low desalination efficiency of the nanofiltration membrane;The microstructure of the spray-dried product was spherical and the surface was uniform.The ?-PL purity and recovery of in the optimized separation and purification process reached 98.7%and 90%,respectively.The plate and frame filtration was used instead of butterfly centrifuge,resulting in the decrease of the water consumption;and 8m3 scale-up production verification test was completed.Research on dynamic structure.The conformation of s-PL in solution and solid powder was studied.Through dynamic light scattering(DLS),the average particle size of ?-PL was as high as 800 nm with pH 8.0.Under normal conditions,?-PL had cationic charges,it interacted with the polysulfone membrane and impurities,was electrostatically adsorbed and accumulated on the membrane surface.Through circular dichroism spectroscopy(CD)and side chain carbamoylation modification,?-PL exhibited an electrostatically expanded conformation in the acidic solution.When the pH of the solution was higher than the isoelectric point,the amino charge disappeared,the formation of intermolecular hydrogen bonds was no longer inhibited,and the conformation of ?-PL changed to the ?-sheet conformation,forming a stable structure.The infrared spectrum fitting calculation showed that part of theconformation of the acidic ?-PL solution was converted into ?-sheet during the spray drying process.Two-dimensional nuclear magnetic resonance showed that under normal conditions,the spacing between ?-PL hydrogen atoms in the solution was greater than 0.5 nm.Research on the antimicrobial activity and mechanism of ?-PL.It was found that freeze-drying could retain its antibacterial activity better than spray drying.The minimum antibacterial concentration of Bacillus subtilis was lmg/mL;?-PL had the strongest activity under neutral conditions,but the change of pH value had no particularly significant effect on the antibacterial activity of ?-PL.Electrostatic adsorption of cations accelerated the binding rate of ?-PL to phospholipids on the surface of the cell membranes during the antibacterial process.Exploration on structural modification.We compared the activity after the alkylation modification with the activity after the carbamoylation modification,revealing that the introduction of hydrophobic groups slightly enhanced the antibacterial activity of ?-PL.The disappearance of the latter activity indicated that the ?-PL main chain might plays a role in the process of combining with the cell membrane to change its permeability.