The Pilot Study On Sea Cucumber I-type Lysozyme Produced By Recombinant Pichia Pastoris

Lysozyme is widely distributed in different organisms,which can destroy the ?-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residuals in bacterial peptidoglycan,thus damaging cell wall and causing cell lysis.Lysozyme,as a natural protein,has characteristics such as non-toxic,easy to absorb and no pollution to human cells.The i-type lysozyme gene was isolated and identified by our laboratory for the first time in 2007,which was expressed in prokaryotic cells and eukaryotic cells respectively.Further studies showed that the i-type lysozyme has broad-spectrum antibacternial activity.Recent progress in genetic engineering makes it possible that some yeast cells have been used as bioreactors to be efficient expression of exogenous genes,especially the Pichia pastoris expression system.The promoter of the alcohol oxidase I(AOX I)gene in P.pastoris could be efficiently induced and strongly initiated.Thus it is suitable for high level expression of exogenous genes.This project is based on these advantages of P.pastoris to produce the i-type lysozyme product by a series of fermentation experiments using shake flask,5 L and 30 L fermenter.In this study,the genetic strain of P.pastoris HS3-1 was used to express the sea cucumber lysozyme protein,which the strain has been constructed previously in our laboratory,First of all,the fermentation conditions of the genetic strain were optimized at the level of shake flask.The optimum fermentation conditions were determined as the initial pH 6.0 and the temperature of 30?.For the induction,1.0%methanol was added to the culture medium every 24 h.The induced time was 96 h.Based on the analysis,the yield of sea cucumber lysozyme in fermentation broth was 5.12 mg/L.The fermentation supermatant was concentrated by ultrafiltration.The target protein was purified by nickel ion affinity chromatography.Finally,the enzyme activity was 868.57 U/mg.The fermentation production of the P.pastoris genetic strain HS3-1 was further carried out using a 5 L fermentor.The yield of sea cucumber lysozyme increased significantly in the fermentation process due to the significant increase of dissolved oxygen(DO).The fermentation medium was basal salt medium,and the fermentation process parameters were controlled at temperature of 30?,pH 6.0,stirring speed 800 r/min and DO 30%.First,the medium was cultured about 24 hours.After the basie glycerol is exhausted,glycerol was added with the rate of 8.15 mL/L/h.When the cell wet weight reached 180 g/L?220 g/L,stop to add glycerol for starving the cells for 1 h,so that the glycerol in the culture is exhausted.Then methanol was added into the culture to induce the lysozyme protein.The concentration of methanol in the culture was controlled at 1%and the induced time was 96 h.The results showed that the enzyme yield of fermentation was 53.26 mg/L.The product was purified by nickel ion affinity chromatography.The enzyme activity was 1238 U/mg.The result of antibacterial experiments showed that the purified sea cucumber lysozyme had significant antibacterial activity against Staphylococcus aureus,Micrococcus lysodeikticus,Vibrio parahaemolyticus and Pseudomonas aeruginosa.On the basis of the fermentation of 5 L fermentor,the scale-up fermentation production of the P.pastoris genetic strain HS3-1 was further carried out using a 30 L fermentor.In the fermentation process due to the increase in the space of the fermentation tank,the fermentation tank can withstand a certain tank pressure,it can reduce the production of foam and reduce the amount of defoamer,thus it can conducive to the late separation and purification.In addition,30 L fermentation has more stability than 5 L fermentation due to the less interference of external environment.The fermentation medium and the fermentation process control were the same as the 5 L fermentor,but the separation and purification processes of the post-treatment were improved.Due to the large amount of fermentation broth by 30 L fermentor obtained,the high-speed tube centrifuge was used for solid-liquid separation.Then the fermentation supermatant was purified and concentrated by 30 kD and 5 kD roll membranes installed in the ultrafiltration membrane equipment.Finally,the purified solution was dried by spray drying to obtain the product of sea cucumber lysozyme.The enzyme activity was 760.5 U/mg.Through the research of this project,the step-by-step amplification of the production of sea cucumber lysozyme by fermentation of P.Pastoris was carried out.The study has provided the theoretical basis and technological parameters for the subsequent industrialized fermentation production,separation,purification,expressed product,and laid the foundation for the application of the sea cucumber lysozyme.