Study On The High-level Expression Of Human Lysozyme And Its Antibacterial Effect

Lysozyme,also known as muramidase,is widely used as an antibacterial agent in the medicine,food,and feed industries.Human-derived lysozyme（HZM2）has high biological activity and thermal stability,and its antibacterial activity is three times that of egg white lysozyme（ELZM）,and is safer and has lower antigenicity.It is expected to be used on a large scale clinically in the future.However,human lysozyme cannot be extracted from the human body on a large scale,so the popularization and use of HZM2 is greatly restricted.The construction of genetically engineered strains expressing recombinant HZM2 is an effective means to solve this problem.In this study,Pichia pastoris X33 was used to express wild-type HZM2,ELZM,T4 phage lysozyme（T4L）,signal peptide and structural modifications were carried out on the plasmid with high antibacterial activity,multi-gene dose expression.Finally,scale up the culture to study the antibacterial activity of the fermentation supernatant.The specific content is as follows:1)The wild-type HZM2,ELZM and T4L recombinant strains were constructed,and after shaking flask fermentation,the optimal antibacterial activity recombinant strain P.pastoris/p PICZα-HZM2-8(2550 U·m L^-1)was optimized by genetic engineering.Modification ofα-signal peptide.First,delete the four amino acids EAEA at the end of the signal peptide,and the enzyme activity of the optimal recombinant strain P.pastoris/p PICZα-d4-HZM2-8 is 3483 U·m L^-1which is 1.37 times that of the wild-type recombinant strain.Secondly,using the natural human type I collagen signal peptide to replace the Pre peptide sequence of theα-signal peptide,the optimal strain P.pastoris/p PICZα-COL1P-d4-HZM2-7 has an enzyme activity of 7403 U·m L^-1,which is 2.13times of the deleted EAEA strain and 2.94 times of the original strain.2)Optimize the structure of HZM2.First,construct a recombinant strain with hydrophobic short peptides added to the C and N ends of HZM2.After the same batch of shake flask fermentation,the enzyme activity is lower than that of HZM2.Secondly,this study was the first to increase the expression efficiency of lysozyme by mutating two Kex2protease action sites in the HZM2 sequence.The recombinant strain P.pastoris/p PICZα-COL1P-d4-HZM2-2KK-3 shake flask enzyme activity was 15379 U·m L^-1.It is 2.07 times of the undeleted Kex2 site and 6.03 times that of the original strain.After the strain was amplified in a 3 L tank,the enzyme activity of the fermentation supernatant could reach 101248 U·m L^-1.3)Construction of a multi-gene dosage plasmid.In this study,the HZM2 gene was integrated by deleting the G418 resistance gene.The results showed that with the increase of the HZM2 gene dose,the enzyme activity and protein content of P.pastoris expressing lysozyme gradually increased.The shake flask fermentation enzyme activity of the recombinant strain with six gene doses was 24533 U·m L^-1,which was 2.3 times the single gene dose,and the protein content of 0.302 mg·m L^-1was 2.9 times the single gene dose.After the strain was enlarged and cultured,the supernatant enzyme activity of the fermentation broth could reach 312360 U·m L^-1,and the protein content was 2.3 mg·m L^-1.4)Antibacterial test of lysozyme.In this study,Escherichia coli,Bacillus subtilis,Staphylococcus aureus,Candida albicans,and Pseudomonas aeruginosa were selected as representative bacteria.The growth curve was drawn and the minimum inhibitory concentration of HZM2 was measured.The antibacterial activity of the fermentation supernatant of the mutant strains,studies have shown that the mutant lysozyme has further enhanced antibacterial activity against Gram-positive bacteria（G⁺）,and has a certain antibacterial effect against Gram-negative bacteria（G^-）.The recombinant fermentation product had an inhibitory effect on all tested bacteria when the enzyme activity was 66667U·m L^-1.