Display Of Mn-SOD On The Cell Surface Of Lactococcus Lactis Using Full-length And Truncated AcmA As The Carrier

Using AcmA(N-Acetylmuraminidase,a major autolysin from Lactococcus lactis) and cA domain(the peptidoglycan-binding domain of its C-terminus) as the anchoring motif,we established two systems for displaying Mn-SOD from E.coli K-12 on the cell-surface of L.lactis ATCC11454 strain to construct the whole-cell biocatalyst.Then we measured the cell-surface display efficiency of two systems.Firstly,L.lastis strains from the fresh milk of the creamery was screened.Using the Gram-positive Staphylococcus aureus as the indictor bacterium,fourteen antibacterial strains were initially obtained by the bilayer-media screening method from the raw milk samples,and one isolate was found to exhibit the higher antibacterial activity against the indicator.This isolate was further studied on its individual and cultural morphology features,partial physiological and biochemical reaction activities,G+C content,the sequence features of the 16S rDNA and the species-specific N-acetylmuraminidase gene(acmA),consequently,it was identified as the L.lactis subp.Lactis strain,named as MB191.An evaluation of the antimicrobial spectra of MB191 subsequently was performed,it showed the remarkable activities against not only the tested Gram-positive bacteria,but also several Gram-negative bacteria including Pseudomonas syringae and P.fluorescens, as well as the yeast Debaryomyces hansenii,which was a distinctive feature that was not reported prior to this study.cA-domain has been confirmed previously the peptidoglycan-binding activity by attaching to the N-acetylmuramic acid.And it has used as an anchoring motif for functional cell surface display systems construction.In this study,acmA gene was amplified from MB191 and sequenced.According to the analysis of the primary structure of AcmA,we engineered the fused gene named as asc,by SOE-PCR,which encoding the signal peptide sequence of AcmA and cA domain.Mn-sodA gene which encoding the superoxide dismutase of E.coli K-12 was amplified,digested and inserted into the E.coli-L.lactis shuttle vector pMG36k,and the expression vector pMB 192 was constructed.Subsequently,asc and acmA gene were digested and ligated into the direct upstream of Mn-sodA gene of pMB 192 respectively for construction of cell-surface display vehicles,that is,pMB193(harboring asc/Mn-sodA fused gene) and pMB194(harboring acmA/Mn-sodA fused gene).They were introduced into L.lactis ATCC11454 by electroporation.The resulting transformants were designated MB192,MB193 and MB194.SDS-PAGE showed that the presence of the fusion protein of Asc-SOD with the expected size of 70kD and AcmA-SOD with theexpected size of 80kD.At last,we measured the SOD activity of MB192,MB193 and MB194 by xanthinoxidase enzymic method,that is,1.53±0.38U/mL,2.63±0.51U/mL and 3.51±0.64U/mL,respectively.And MB194 had the optium cell surface display efficiency,up to 56.4%.