Recombinant Expression And Activity-enhancing Of Sus Scrofa Lysozyme

With the increasing frequency of food safety incidents and the abuse of feed antibiotics,to find a safe,pollution-free,highly efficient and stable,broad-spectrum antibacterial replacer of feed antibiotics which could be produced in large scale is imperative.As a part of the immune system of animal bodies,lysozyme is a natural replacer of antibiotics,and it has been used in medicine,biological research,food and feed industry with a bright future.However,as one of the most important members of animal husbandry,pig has not yet had a special lysozyme to use.Fortunately,as a natural important protective screen against bacteriosis in the body of pig,Sus scrofa lysozyme(SSL)is resistant to pepsin,suggesting it a good candidate to replace feed antibiotics used in pig.However,due to a shortage of source,as well as its little resistance to gram-negative bacteria,SSL has not been reported to be used.In this study,gene engineering and microbial fermentation were employed to produce SSL in scale.At the same time,SSL was hydrolyzed by trypsin and two anti-gram-negative bacteria peptides(LP and SP)were obtained by purification of gel filtration,reverse phase chromatography and liquid chromatography-mass spectrometry(LC-MS).Then,gene modification method was used to improve the action effect of antibacterial peptide SP,and a novel SSL which is resistant to both gram-positive and gram-negative bacteria was obtained.In addition,the action mechanism of the novel SSL was studied,laying a theoretical foundation for its further application.(1)According to the gene reported in NCBI database,the coding sequence of SSL was synthesized and optimized based on the codon preference of host E.coli.The genes was digested by enzymes BamH ?and Hind ?,connected with vector pET-28a(+),transformed into host E.coli BL21(DE3),and was successfully expressed.Under the optimized condition,the recombinant host was firstly incubated at 37? for 3 h,and then induced by 0.8 mmol·L-1IPTG at 25? for 8 hours.By the process of washing,dissolution,unfolding and concentration,bio-active SSL with concentration of 166.91±3.37 mg·L-1and specific activity of7950.42±226.67 U·mg-1 was obtained.Moreover,SSL was also expressed by Pichia pastoris system using X-33(pPICZ?A),within which the effect of two methods for codon optimization to SSL expression were studied.It was found that the method of “random optimization” has an obvious improving effect of SSL expression in P.pastoris,in which a production of 189.28±4.16 mg·L-1 and a specific activity of 2845.38±124.19 U·mg-1 was finally obtained,with a 2.6 fold expression quantity of original SSL gene.The method of “one amino acid one codon” has no effect on the improving production of SSL in P.pastoris X-33 in this study.Furthermore,the expression levels of SSL using constitutive expression vector pGAPZ?A and protease-deficient host SMD1168 H were both not as effective as X-33(pPICZ?A).By the process of affinity chromatography or the unfolding of SSL,combining the purification of ultrafiltration.SSL from E.coli or P.pastoris of electrophoretic pure were obtained.With the study of enzymatic properties,they were found to have the same optimal reaction temperature of 35 ? and the same optimal reaction pH of 6.0,which were alsoconsistent to their theoretical values.The results suggested that production of SSL by fermentation was feasible.Comparing the processes of SSL production by E.coli and P.pastoris system,it was found that E.coli system had a higher specific activity,a shorter production period and a better preparation for downstream process.As a result,E.coli system was selected for further research in this study.(2)With the process of SSL hydrolyzation by different proteases,SDS-PAGE analysis and the determination of antibacterial activities of the mixtures,SSL was found to be resistant to pepsin.Among all experimental samples,trypsin hydrolysate had a highest antibacterial activity of 2.81 against E.coli,with a sterilizing rate of more than 99%.By the means of gel filtration,reverse phase chromatography and LC-MS,two anti-gram-negative bacteria peptides(LP and SP)were obtained,with amino acid sequences of G-V-S-L-A-N-W-V-C-L-A-K(LP)and A-W-V-A-W-K(SP),respectively.These two peptides were then synthesized and analyzed for antibacterial spectrum.LP was found to be anti-gram-negative bacteria,but have no effect on gram-positive ones.Distinctly,SP was found to be able to kill both gram-negative and gram-positive bacteria.With the help of circular dichroism(CD)analysis,Swiss-modeling and atomic force microscope detection,both of the peptides were speculated to kill bacteria by penetrating the cell membrane of target cells,similar to the way of peptides with helix-loop-helix structure.However,the specific action models of these peptides were different: LP was thought to act as a carpet model,while SP was considered to form a hole in the cell membrane with a aid of its ?-helix structure.(3)Fusing the coding sequences of the better peptide SP in antibacterial activity,the coding sequences of the helix-loop-helix structure in SSL containing SP,and six copies of coding sequences of SP to N-terminal or C-terminal of coding sequence of SSL,respectively.The constructed genes were expressed in E.coli BL21(DE3),and some fused SSLs with significantly improved antibacterial activity against gram-negative bacteria were obtained.Under the same conditions,N-terminal ones showed higher antibacterial activities than C-terminal ones.With the aid of CD analysis and Swiss-modeling software,N-terminal of fused SSLs was found to be closer to the active cleft of SSL,which was thought to be important in combing to its acting substrates.Among all fused SSLs,N-terminal fusion product 6SP-SSL showed a highest antibacterial activity.The combination of 6SP to SSL,on one side,improved the rate of SP contacting with acting substrates;one the other side,6SP conferred a higher hydrophobicity,which was thought to beneficial for SSL combining its active sites of the target cells.In addition,the synergistic effect of 6SP and SSL was discovered,especially when Staphylococcus aureus ATCC 25923 and E.coli ATCC 25922 were treated.The production of 6SP-SSL provided a feasible,high effective,and wider spectrum way in improving the antibacterial activity especially against gram-negative bacteria for C-type lysozyme.(4)By atom force microscope scanning and the monitoring of membrane potential of target cells of E.coli ATCC 10798,6SP-SSL was found to be able to destroy the integrity of the cell wall,causing a change of cell permeability and the outflow of insoluble substance,resulting in the death of target cells.The phenomenon was consistent with the actionmechanisms of SSL and SP.As a result,we speculated that when 6SP-SSL contacted with target cells,SP firstly form a hole in the cell membrane by its ?-helix structure or hydrophobic interaction,then SSL was able to combine with its acting substrates and they worked together.Moreover,with the aid of flow cytometry monitoring,programmed cell death of partial target cells treated by 6SP-SSL were detected.In fact,the rate of cells with programmed cell death was dependent on the concentration and treating time of 6SP-SSL.The result suggested that not only could 6SP-SSL kill target cells by direct sterilization mechanism,but also it was able to induce the programmed cell death of treated microbes,causing an indirect sterilizing effect.A further research showed that 6SP-SSL could reduce the expression level of maz E by turn down the transcriptional level of its coding gene,resulting in an accumulation of the toxin maz F in the toxin-antitoxin system of target cells,which could induce the programmed cell death of host.