Expression Of Human-Derived Antimicrobial Peptide LL-37 In Pichia Pastoris And Its Application In Milk And Juice

Research shows that that antimicrobial peptide(AMP)has a wide variety of sources and is commonly found in nature.AMP has a broad spectrum of antibacterial activity and high bactericidal ability.Its molecular weight is small,easy to be metabolized,and not easy to produce drug resistance with fast bacterial inhibition,which has stable structure,good water solubility.its α-helical structure in the orderly arrangement in water can enhance the stability of protein structure,and minimize protein degradation,which will have unlimited development prospects in the new natural antiseptic and new medical field.LL-37 is the only antimicrobial peptide from the cathelicidins family in human body,named LL-37 because of its 37 amino acids and the first two amino acids are Leucine(L),with a molecular weight of about 4.5 KD.It is a class of cationic peptides that playing an important role in human immune response.However,the high cost,low yield and low activity of the antimicrobial peptides obtained by chemical synthesis have limited the large-scale application of LL-37.Pichia pastoris SMD1168 H is a protease gene deletion strain,and using it as a genetically engineered host strain can reduce the hydrolysis of proteins,and using the yeast system to express LL-37 can be secreted directly to the extracellular,simplifying the subsequent purification process.Methods.In this study,the optimal expression conditions of recombinant strain were obtained by single-factor test and orthogonal test method after obtaining positive clones;ion exchange chromatography was used to purify LL-37 expressed by the engineered strain,and high performance liquid chromatography and mass spectrometry were used to identify the expressed product LL-37;Staphylococcus aureus and Escherichia coli were used as test organisms to determine the minimum inhibitory concentration,and LL-37 was applied to freshly squeezed juice and milk for preliminary application of preservative finally.The results indicate.(1)The optimal medium are as follows: the ratio of nitrogen source to carbon source is4:1,and the ratio of yeast powder to ammonium sulfate in nitrogen source is 2:1;the optimal expression conditions are as follows:the expression temperature is 28℃,the incubation time is 36 h,the initial amount of strain transferred during expression is 2m L/25 m L,and the shaking flask speed is 200rpm/min.The final expression amount of LL-37 could reach 79% of the total protein in the supernatant.(2)After the supernatant of the fermentation was concentrated by ultrafiltration with3000 D ultrafiltration membrane and further impurity removed by dialysis bag with 1000 D,the small molecules lower than 4.5KD were basically removed;Anion exchange chromatography column(Q column)is used for purification and impurity removal.The Q column can combine most of the negatively charged impurity proteins in the supernatant,and the purity can reach 80% through high performance liquid chromatography.(3)According to the bacteriostatic activity test,the minimum bacteriostatic activity of LL-37 against Escherichia coli is 51.2ug/m L and that of Staphylococcus aureus is 40ug/m L;the application of LL-37 to milk and freshly squeezed juice can significantly inhibit the growth of microorganisms,laying the foundation for the initial application of LL-37 in freshly squeezed juice.