Study On Recombinant Expression,Optimization Of Fermentation Parameters,Purification And Antibacterial Mechanism Of Ruminant Stomach Lysozymes

To utilize yeast strains to recombinantly express ruminant stomach lysozyme,the lysozyme genes of ruminants(such as yak,Tibetan sheep,Tibetan goat)were used as research objects in this study;and the yeast strain capable of expressing recombinant stomach lysozyme was constructed and screened,finally,more than 60 recombinant yeast strains were obtained.The SDS-PAGE electrophoresis results of the fermentation supernatant showed that the molecular weight was 15 KDa,which was close to the theoretical value.The enzyme activity of recombinant bovine stomach lysozyme(rbs LYZ)and recombinant Tibetan goat stomach lysozyme(rgs LYZ)in the fermentation supernatant was higher than that of other ruminant stomach lysozymes.The recombinant Pichia pastoris strain(G6-1)was used as the research object to study the effects of fermentation parameters and gene copy number on the expression of recombinant bovine stomach lysozyme.(1)The factor screening test design(Plackett-Burman)screened the fermentation parameters,and found that the fermentation parameters(culture volume,induction time,methanol concentration and Inoculation volume)significantly affected the expression of recombinant bovine stomach lysozyme(P<0.05).(2)The response surface methodology was used to study the effects of fermentation parameters such as culture volume on the activity of recombinant bovine stomach lysozyme.It was found that when the culture volume was 42 m L,the induction time was 89 h,and the methanol concentration was 1.1%,the activity of recombinant bovine stomach lysozyme was the highest(415.2 U/m L),which was about 1.1 times higher than that of recombinant bovine stomach lysozyme before optimization.(3)The q PCR method was used to study the effect of gene copy number on rbs LYZ activity,it was found that the activity of rbs LYZ increased with the increase of bs LYZ gene copy number in recombinant yeast strains.(4)Compared with the negative control,the recombinant bovine stomach lysozyme has better antibacterial effect on Micrococcus luteus,Staphylococcus aureus and Micrococcus lysodeikus;while it has no inhibitory effect on Pseudomonas aeruginosa,Bacillus cereus and Escherichia coli.Yarrow lipolytica(Polh strain)system was used to recombinantly express the yak stomach lysozyme.The results showed that the molecular weight of recombinant yak stomach lysozyme(recombinant yak stomach lysozyme 1a,rys LYZ1a)was about 15 KDa;the expression of rys LYZ1 a was highest when cultured for 72 hours;The electrophoresis pure recombinant yak stomach lysozyme 1a(rys LYZ1a)was obtained by CM-Sepharose FF column chromatography and other purification methods.The specific activity was determined to be 1855.42 U/mg.It was observed under electron microscope that rys LYZ1 a exerted antibacterial effect by breaking the cell wall of S.aureus;rys LYZ1 a can effectively resist the degradation of pepsin,and it remains partially intact after being treated for 4 h in simulated pig stomach fluid.This study laid the foundation for the widespread application of the plateau ruminants stomach lysozyme.