Effect On Nisin Production By Expressing Nisin Resistance Protein Mutant

The antimicrobial peptide, nisin,produced by several strains of Lactococcus lactis ,which belongs to the Class I bacteriocins called lantibiotics ,is a small (3.4 kDa ) -34-amino acid ,cationic peptide. It kills a broad range of Gram-positive bacteria including important food spoilage and pathogens and relevant clinical bacteria effectively and nisin is safe to human being for it can be degraded byα-chymotrypsin and becomes the only large-scale commercial application of highly efficient natural food preservatives.In nisin-producing L. lactis, nisin resistance/immunity system consists of the lipoprotein NisI and the ABC transporter complex NisFEG.. However, in some nisin non-producing L. lactis strains, nisin resistance could be conferred by a specific nisin resistance gene (nsr) which encodes a 35 kDa nisin resistance protein (NSR). In this study,we adopted genetic manipulation to improve nisin production by introducing nisin resistance protein mutant into nisin producing strains. The results were as follows:1) Site-directed mutagenesis of Ser236 in NSR: conserved domain analysis reveals the presence of a C-terminal conserved tail–specific protease domain (TSPc) at C-terminal of NSR.The site of ser236 of NSR is conserved as all other known tail-specific proteases. Therefore site-directed mutagenesis of Ser236 in NSR was carried out.2) The physiological roles of NSRS236A: in order to study physiological roles of NSRS236A, the recombinant Lactococcus lactis MG1363 strains expressing NSRS236A was constructed and Western Blotting hybridization showed that NSRS236A was localized on the membrane of recombinant expression cells. Peptide release assays suggested that mutagenesis of this Ser to Ala ablated NSR activity of nisin cleavage and SPR analysis revealed that NSRS236A can bind to nisin.3) Effect on nisin production by expressing nsrS236A gene in nisin producing strains: using the trait of NSRS236A can not degrade nisin but can combind nisin, Nisin resistance gene nsrS236A with a strong promoter P59 were cloned into vector pHJ201, then introduced into Lacotococcus lactis NZ9800. In comparison with the control, The expression of nsrS36A had no significant difference in growth rate between recombinant strain and contrast strain. However, it promoted recombinant strain tolerance 40% higer nisin resistance level and stronger antibacterial activity against M. flavus NCIB 8166, which was increased by 1.9 times and 27% when fermented for 6 and 8 hours, respectively.This study showed: NSRS236A , which located in cell membrane, can prevented nisin producting strains by combinding nisin molecular, and improved the nisin resistance level and expression level of strain.Conclusively, NSRS236A were expressed successfully in L.lactis and enhanced nisin resistance level effectively and resulted in improvement of nisin production. The research suggested that nisin yield from the producing strains could be further increased by genetic manipulation and approaches can be used successfully to improve nisin production.