Development Of Microreactor For One-step Separation And Purification Of Protein For Fermentation Engineering

The separation and purification of protein plays an important role in fermentation engineering.The key of this technology is to keep the protein activity.Both of the upstream and downstream face the risk of inactivation: in the process of upstream,the yeast cells not only secrete protein,but also secrete proteases,resulting in the degradation of target proteins.Proteins are sensitive to temperature,p H,organic reagent and other factors during downstream process:separation and purification of protein.Traditional chromatography(such as ion exchange chromatography,gel filtration,affinity chromatography,etc.)have the problems of complex processes and strong non-specific adsorption.However,regardless of chromatographic methods,stationary phase plays an important role in protein separation.so it’s urgent to develop materials that can interact with target protein to separate target protein rapidly and ensure the activity of protein.Metal chelate affinity chromatography(IMAC)is the most widely used method for protein separation and purification currently.The advantages of IMAC are large adsorption capacity,firm binding capacity and wide rage of applications.However,the stationary phase modified by ligands is usually limited by particle size and surface modification,resulting in low group density and low separation efficiency.In addition,affinity chromatography can only be used in the process of post-fermentation,which greatly limits its application fields.In order to solve this problem,PAM-NTA hydrogel composed of AM and NTA is prepared by photocrosslinking.NTA is the affinity ligand which used for protein separation and purification.Cell loaded PAM-NTA microgel was prepared by microfluidic technique to support yeast cells proliferation and protein secretion.During the yeast culture,NTA component plays the role of protein adsorption.At the end of culture process,we capture purified protein through simple centrifugal.It’s worth mentioning that we realize the integration of fermentation and separation.Firstly,the selection of materials and synthetic methods of NTA hydrogel for protein separation and purification were explored.(1)The first material we tried is sodium alginate which is one of the most commonly used biocompatible materials for amino grafting.The purification performance of Alg-NTA product is compared with that of commercial agaroseNTA(Ni-NTA QZT 6FF).The result show that NTA ligand density of grafted product is only 0.458% of commercial Ni-NTA QZT 6FF.Subsequently,NTA is grafted with double bonds by the reaction of succinimide and amino formd amide bonds,then copolymerized with photocrosslinked monomers to form hydrogel for separation and purification of protein.Then it was photocrosslinked with PEGDA/AM,and the results showed that AM-NTA hydrogel has a better ability to recover target protein,and the optimal concentration of imidazole is 200 m M.(2)Furthermore,the difference of gel-forming performance,Ni2+ binding amount,swelling property and porosity rheology of AM/NTA hydrogel with different proportion of two components was investigated.When the molar concentration(M)was constant,with the increase of NTA components,the gel-forming performance decreased,the storage modulus G ’decreased,and the density of hydrogel network became more loose.The Ni2+ binding amount of AM-NTA hydrogel increased with the increase of NTA components.When the volume of AM-NTA hydrogel was constant,the Ni2+ binding amount of AM-NTA hydrogel in groups 4-1:2(M=4,AM:NTA=1:2)was the highest,reaching 32.53±0.03μmol/m L.Yeast cells survive the harsh conditions of encapsulating by photo-crosslinking and protein secretion within AM-NTA microspheres were verified.Remarkably,the target protein eluted from microspheres retains its natural activity with high purity compared to traditional motheds.(1)The most suitable condition to fabricate PAM-NTA hydrogels for cell proliferation was explored including UV illumination time,total molar concentration(M),proportion of composition of AM/NTA hydrogel and amount of crosslinking agent(MBAA),which co-regulate the porosity of AM/NTA hydrogel.Results show that optimum condition is UV illumination time=30s,MBA =0.4% total molar concentration,AM/NTA hydrogel is group 3-3(M = 3,NTA/AM =1:2).(2)Cell proliferation and protein secretion were further developed by loading agarose microparticles into AM-NTA hydrogel.The activity of yeast cells within microspheres were verified by live/dead assay.And the target protein eluted from microspheres directly was characterized by SDS-PAGE gel electrophoresis.When the ratio of agarose microparticles to 4-3 AM-NTA hydrogel was 1:1,the cell activity was optimum and the cell proliferation continue rising in 3 days.The concentation of target protein collected from microspheres was 0.05±0.03 mg/m L,and its purity was higher than that of commercial agaorse-NTA filler.To sum up,the AM/NTA hydrogel for separation and purification of protein was investigated by encapsulating yeast cells within microspheres to capture target protein with high purify effectively,and eases of purification with natural activity.This work provides a dependable strategy for protecting target protein form proteases to retain natural activity and achieving separation and purification of target protein from fermentation broth directly.