High Expression In Pichia Pastoris And Modification Of Codon-optimization Egg White Lysozyme

Lysozyme(LYZ)is a kind of natural antibacterial substance widely existing in microorganisms,animals,plants and human body.It can dissolve mucopolysaccharide in the cell wall of pathogenic bacteria nonspecifically.Lysozyme can replace antibiotics to reduce the harm caused by bacterial resistance.It has a good application prospect in food,cosmetics,drugs and so on.It mainly caused the breakdown of the β-1,4-glycoside bond between N-acetylparitic acid and N-acetylglucosamine of the cell wall,leading to the death of bacterial.The main target of lysozyme is gram-positive bacteria with thick peptidoglycan layer.At present,the commercial lysozyme was mainly extracted from egg white.It limits by sources,low purity of chemical extraction and great loss.Meanwhile,as a kind of non-broad-spectrum antibacterial agent,the application of lysozyme is also limited.Objective: In this study,pichia pastoris expression system was selected to express egg white lysozyme with higher yield.In order to broaden the antibacterial spectrum of egg white lysozyme protein was modified by biological method.It helps for expanding the market and application of egg white lysozyme and also provides theoretical reference for the molecular modification of protein.Methods: The strategy of codon optimization was adopted to obtain a yeast strain which could express egg white lysozyme efficiently.Firstly,ORF sequence(Gene ID: 396218)of lysozyme was partially replaced according to the codon preference of yeast to obtain a new optimized Gene more suitable for yeast expression system.Through Infusion cloning enzyme,the optimized gene was connected to the secretion of expression vector p PIC9 K cut by NotⅠand Eco RⅠ.Recombinant plasmid p PIC9K-co EWL was constructed.After sequencing,linear plasmid by SalⅠ was transferred into yeast cells GS115.After screening through MD plate and G418 resistant plate,a yeast recombinant(G-p-co EWL)was obtained.Fermentation was induced with 1% methanol at 28℃,p H 6.0,rotation speed of 240 rpm.Compared with Gram-positive bacteria,there is less peptidoglycan and more lipids in the cell wall of the negative bacteria.This difference may be the reason why the egg white lysozyme has little resistance to G-.To broaden the antibacterial spectrum,four different hydrophobic short peptides were added at the end of egg white lysozyme.They were Val-Val-Tyr-Pro derived from soybean,Ile-Ile-Ala-Glu-Lys from β-lactoprotease,Asn-Pro-Val-Trp-Lys-Arg-Lys from Spirulina extract and Phe-Peu-Leu-Leu-Pro-His from hazelnut.These short peptides are reported in the literature that played a role in the process of reducing fat.Firstly,according to the codon preference,the sequence of the short peptide was translatedand sequence of the three peptide and five peptide was linked to the 3’ end of the target gene by PCR through primer.The longest gene sequence was synthesized by the company.Similarly,the four genes of interest were ligated to the p PIC9 K vector.After digesting by Sal Ⅰ,liner plasmid was transferred into Pichia pastoris cells.Four strains of yeast recombinants were obtained after resistance screening.And the new egg white lysozyme with short peptide was obtained by fermentation at 28 ° C,p H 6.0,rotation speed of 240 rpm with 1% methanol.Four G-bacteria and three G+ bacteria were used as the testing bacteria to evaluate the antibacterial spectrum of new lysozyme.Results: A yeast transformant which could grow in the presence of 15 mg/ m L G418 was obtained after screening and confirmed by colony PCR.At 28 °C,p H6.0,rotation speed of 240 rpm and methanol concentration of 1%,the culture of G-p-co EWL was carried out for 72 hours.The protein fraction of the bottle shake fermentation supernatant was 607 mg/L,and the enzyme activity reached 677 U/m L.The sequencing results showed that the four short peptide sequences were successfully ligated to the end of co EWL.After transfering into yeast,the yeast recombinant were obtained and expressed four new lysozymes of CH1,CH2,CH3 and CH4 successfully.The SDS-PAGE results showed the target protein bands.The results of bacteriostatic test showed that the fermentation supernatant showed stronger resistance to Gram-negative bacteria than the standard lysozyme,and antibacterial activity of CH4 was most obvious.Meanwhile they maintainted the resistance to Gram-positive bacteria.