Purification Of Melittin Based On Silica Nanoparticles

Melittin,the major active ingredient in bee venom,has high biological activity and a variety of pharmacological activities.In addition,melittin is often used as a model peptide to study the interaction between antimicrobial peptides and biofilms.At present,melittin is mainly isolated and purified from crude bee venom.It is still a challenge to develop separation and purification method which is easy to operate at low cost and can ensure its purity and activity,which limits the wide application of melittin.In recent years,particles with unique shape and large specific surface area have been widely used as synthetic adsorbents in the fields of bioengineering and biotechnology.The exploration of melittin purification by synthetic particles has been reported and a certain purification effect has been achieved.As a material that is simple to synthesize and easy to be surface-modified,nanosilica particles are widely used in the field of adsorption.However,there is no report for adsorbing melittin.In this paper,nano-sized silica particles were synthesized by St（?）ber method and characterized by transmission electron microscopy.The particles were uniform and well dispersed with an average particle size of86±10 nm.The surface of the silica nanoparticles was alkylation-modified and characterized by FT-IR.The infrared spectrum was consistent with the standard silica spectrum.The alkylated modified spectrum showed new peaks representing-CH₂-antisymmetric and symmetric stretching vibration,it was demonstrated that the alkyl chain was successfully grafted onto the surface of the silica nanoparticles.In the adsorption test section,the effects of time,pH and melittin concentration on the adsorption behavior were investigated.The adsorption time was determined to be 20 min;the adsorption amount of MEL increased first and then decrease with the addition of pH,and the maximum adsorption amount（55.28μg/mg）was obtained at the p I of melittin.The results of the kinetic equation fitting show that the adsorption system is more consistent with the second-order kinetic model,indicating that chemisorption may be the rate-limiting step of the adsorption rate.The change of Zeta electric potential on the surface of the nanoparticles before and after adsorption indicates that MEL is bound to the surface of the nanoparticles during the adsorption process.When the adsorption is completed,according to the results of the adsorption amount with the change of pH and melittin concentration,the melittin is desorbed by the method of ultrasonic solvent redispersion after centrifugation,and the recovery reaches 92%.The desorbed MEL solution was subjected to SDS-PAGE electrophoresis.Compared with melittin standards and BV,it is proved that AM-NSP has significant purification effect on melittin.In addition,it was also confirmed that AM-NSP has a significant purification effect on MEL in BV by comparing the BV and desorption MEL in chromatograms of HPLC.The hemolysis test,compared with the melittin standard,proved that the purified MEL maintained hemolytic activity.The adsorbent was continuously reused 5 times,and the recovery did not change significantly.This thesis attempts to purify melittin by surface-modified nano-silica for the first time,and obtains a good purification effect,which provides a new idea for the purification of melittin.