Heterologous Expression,Optimizing The Expression Condition Of The Lysostaphin And Fowlicidin-1 In Kluyveromyces Lactis And Pichia Pastoris

Recently,the emergence of all kinds of drug-resistant strains leads to increase the difficulty of disease treatment,due to the overuse of antibiotics.These drug-resistant strains threaten our human beings health severely.It is important to hunt for antimicrobial substance of safety,effectivity and non-drug-resistant as for the replacement drugs of antibiotics.Lysostaphin can treat infection by the Staphylococcus aureus efficiently,and it can not cause the emergence of drug-resistant strains.Antimicrobial peptide has the characterization of small molecular and good stability.It does not generate drug resistance by the way of physical sterilization,so it has enormous application potential.In this paper,the Lysostaphin and Fowlicidin-1 were expressed by using recombinant gene technology.The main results were as follows.?1?According to the sequence of lysostaphin gene from Staphylococcus simulans and codon bias of Kluyveromyces lactis,the PCR primer was designed to amplify the lysostaphin gene.The gene was inserted in pKLAC1,and transformed to K.lactis GG799.The K.lactis GG799/pKLAC1-Lys was cultivated to express Lys.?2?We obtained a high expression strain?mu4#?by using powerful mutagen?N-methy1-N-nitro-N-nitrosoguanidine,NTG?on the recombinant and optimized the expression condition.The fermentation broth of mu4#was purified by Ni-NTA agarose and the enzyme characterization was studied.The result showed that the activity of Lys was approximately 5.2times?8000U/L?higher in the mutation.The optimal inoculum dose of the mutant?mu4#?was40g/L;Galactose and NH₄NO₃?20g/L?were added in every 24 hours,Lys exhibited optimal expression at pH 7.0-7.5;Furthermore,the Lys enzyme optimal reaction performed at pH 7.0-8.0and temperature at 37°C.The recombinant Lys was stable below 40°C and pH between 3.0 and6.0.Sr²⁺stimulated its activity whereas Ba²⁺?Ca²⁺?Zn²⁺?Cu²⁺?Mn²⁺?Mg²⁺inhibited the activities.This research accomplished Lys recombinant expression,yield improvement by chemical mutagenesis in K.lactis and characterization of lysostaphin.These research results provide profound guiding significance for the large-scale production and application of recombinant lysostaphin.?3?Under the SEM,we observed the effect that lysostaphin sterilized the Staphylococcus aureus.It decreased the concentration of the Staphylococcus aureus.The volume of cells diminished and a number of cells bursted.We used mu4#strain do pilot scale test in a 100L fermentor.The result showed that the activity of Lys was approximately 45 times higher in the fermentor.The biomass added up to 210g/L.?4?We constructed the high expression strain SMD1168/Ppic9k-F3 by the expression system of Pichia pastoris.MS experiment demonstrated that the strain SMD1168/Ppic9k-F3 successfully expressed Fowlicidin-1.The Fowlicidin-1 has good heat stability.The Fowlicidin-1 showed good antibacterial effect on Staphylococcus aureus,escherichia coli and clostridium perfringens,but without antibacterial effect to lactobacillus in the condition of approaching alkalinity.