The Antimicrobial Characteristics And Mechanism Of Recombinant Mytichitin-CB Produced By Pichia Pastoris Expression System

It is an effective way to find new antimicrobial agents that can solve the problem of bacterial resistance.The emergence of antimicrobial peptides has become one of the most promising new antimicrobial agents.It has many advantages:wide antibacterial spectrum and high thermal stability.The antibacterial peptide,Mytichitin-CB,was isolated and identified from the Marine mussels.It contains 55 amino acids,and these six cysteines can form intra-molecular disulfide bond.Forthermore,the molecular structure was stable,antimicrobial property was strong and the antibiotic spectrum was wide.Therefore,it had great applicatial values.In this study,a new antimicrobial peptide,rMytichitin-CB was expressed in the expression system of pichia pastor is.The expression of the target protein was as high as 52.8 μg/mL when the final concentration of methanol was 2%and the induction time was 120 h.The supernatant of the fermentation broth was purified by the affinity of nickel column,and the rMytichitin-CB was obtained successfully.The antibacterial activity experiment revealed that rMytichitin-CB had good antibacterial activity against 9 kinds of standard strains and 13 kinds of drug-resistant strains.However,antibacterial kinetics studies showed that rMytichitin-CB had a little antibacterial effect on S.aureus.These sdudies showed that rMytichitin-CB had a strong antibacterial activity rather than bactericidal activity.The temperature stability study results indicated that the rMytichitin-CB could keep good stability in 4℃,15℃,37℃,65℃ and 90 ℃,respectively.The results of acid-base stability study showed that rMytichitin-CB could maintain good stability under the conditions of PH 2,4,6,8 and 10,respectively.The results of enzyme stability showed that rMytichitin-CB had good stability in pepsin,protease K and trypsin,respectively;but it was sensitive to papain.The study results shown that rMytichitin-CB had low hemolysis activity to red blood cells of mouse and human,and in certain degree had cytotoxicity to the Human Embryonic Kidney cells(HEK293)and the Baby Hamster Syrian Kidney cells(BHK21).There was only one amino acid Mytichitin-CB different from Mytichitin-A at the 39th place,but the activity rMytichitin-CB was higher than that of rMytichitin-A.Therefore,the histidine in the 39th place of rMytichitin-CB was changed to tryptophan,lysine,isoleucine and alanine,and the mutants were H39W,H39K,H39I,H39A,respectively.The results of antibacterial activity were shown as follows:the wild type had better antibacterial activity than these mutants,and the antibacterial activity of H39W was the best in these mutants,and H39K followed the H39W,and H39I and H39A was the most weak.Based on the above studies,we hypothesized that the imidazole ring of histidine played an important regulatory role in the maintenance of protein conformation.The rMytichitin-CB was then tested in the cell membrane,which showed that antimicrobial peptides could destroy the cell membrane of S.aureus.