Study On The Construction And Activity Of Phage Fusion Lysin

Phage lyase is a kind of cell wall hydrolytic lyase expressed by phages in late stage of bacterial infection.It shows advantages of non-proliferation,easy directional operation and no(less)bacterial resistance effect,but also disadvantages of narrow cleavage spectrum and poor thermal stability.Despite compound enzyme preparation can offset a series of defects of phage lyase,different enzymes need to be produced separately and then mixed in proportion.This operation raises labour and material costs.Via fusion of two lyases using genetic engineering technology,the advantages of different lyases can be further incorporated,so as to obtain a new fusion lyase that can better overcome the defects.In this paper,lyase gene MMPphg of Meiothermus TG07 phage MMP7 and TSP4 lyase gene TSPphg of Thermus TC16 were used as materials.These two genes were linked using two molecular biology-based methods.Then,a fragment fused by the two genes was cloned in an expression vector to obtain the system with ability to express the fusion lyase.In brief,the two methods of ligation were described as follows:first,no linker was added between the two lyase genes.Their connectors were directly cloned in the same vector after head-to-tail connection,and then constructed into the expression vector by traditional enzymatic ligation to obtain the fusion lyase gene TPIphg,which is then transferred into Escherichia coli(strain BL21(DE3)).Alternatively,a linker was added between these two lyase genes,the two lyases genes are connected by a linker,in the design of primers,the linker peptide is directly added to the primer.Linker was linked to the lyase gene by PCR,and then the two lyase genes were connected by overlapping PCR technology to obtain the fusion enzyme gene ILTphg.After that,it was constructed into the vector and transferred into Escherichia coli(BL21(DE3))for expression and purification,and its enzymatic properties were studied.The experimental results showed that the fusion lyase TPIphg(without linker)could not form soluble protein,and detection of its lyase activities was not further conducted.The fusion lyase ILTphg(with linker)could be well expressed.After exploration of induction conditions,the optimal that was obtained:37?,150 rpm,and 6 h.A final concentration of inductor was 1 g/L,and expression levels of ILTphg with linker was stable in shaking bottle,reaching 60 pmol/m L.The optimal temperature range from 35 to 40?,and that of ILTphg range from 35 to 40?and7 to 8.Na⁺,K⁺,Mg²⁺and other ions activated ILTphg to some extent,while Ca²⁺and Fe²⁺inhibited lyases.Mn²⁺caused occurrence of target protein denaturation.In the experiments that verified bactericidal activities of the fusion lyase ILTphg,it was found that the fusion enzyme ILTphg had obvious cleavage effect on TC16and TG07,which were host bacteria of tspphg-derived phage TSP4 and mmpphg-derived phage MMP07,respectively.When the concentartion of 40pmol/m L of the lyase ILTphg,Staphylococcus aureus,Salmonella and Escherichia coli showed a better inhibitory effect compared with the lytic lyases(MMPphg and TSPphg).When the concentartion of 40 pmol/m L of the lyase ILTphg,the fusion lyase ILTphg presented an anti-bacterial action after the reaction of 10 minshorter than that of the lyase MMPphg and TSPphg.When the concentration of 25 pmol/m L of the fusion lyase ILTphg,a better antibacterial effect than the lyase TSPphg and MMPphg of 40 pmol/m L concentration was exhibited.To sum up,the fusion lyase ILTphg obtained by MMPphg and TSPphg through connection peptides is a soluble protein with high expression level.Compared with the two single lyases before fusion,the enzymatic properties of the fusion lyase ILTphg have not changed significantly,and the lyase activity of the fusion lyase ILTphg has been significantly improved compared with both TSPphg and MMPphg.The action time of fusion lyase ILTphg was shorter,the effective inhibition concentration was lower,and the inhibition effect was more prominent.However,the cleavage spectrum and antibacterial activity of the fusion lyase ILTphg need to be further improved.In conclusion,a series of enzymatic properties and bacteriostatic properties of fusion lyase ILTphg were improved compared with MMPphg and TSPphg phage lyase alone,indicating that this study has certain reference value for improving the enzymatic characteristics,bacteriostatic ability and production performance of phage lyase,laying a foundation for the subsequent development and application.