| Nisin, a 3.5kD antibacterial peptide, is widely utilized in food industry as highly effective, safe and natural food preservative. It contains cyclic lanthionine rings and unusual dehydrated amino acids, which are formed by posttranslational modification. The strucure of nisin is usually used to study on the relationship of structure-function. And, its particular regulation system has received significant attention. Recently, researchers try to make structural variants of nisin by mutagenesis, which would be used to provide information about the structural basis of their physical, chemical and biological properties. In this paper we cloned nisin biosynthesis gene cluster and systematically studied on structure-function relationship ofnisZ promoter and structural gene.1. A total DNA library ofL.lactis AL2 with high yield of nisinZ has been successfully constructed, we has selected the entire nisin biosynthesis gene cluster, and the encoded structural gene was cloned and expressed in L.lactis NZ9800. Sequencing of DNA had been identified that L.lactis AL2 applied to industrialization produced nisinZ, nisZ promoter and structural gene ofL.lactis AL2 was similar to that of current L.lactis.2. The two mutants, which were the combination of mutants with some positive results as described previously by site-directed mutagenesis were applied to a CM-sephadex CM-25 cation-exchange columnby using a FPLC system and then the concentrated fractions applied onto a Sephedax G-25 column for gel filtration. The results showed the antimicrobial activities of two purified nisinZ species decreased than that of wild-type nisinZ, their spectrum of antimicrobial activities and their solubilities were similar to nisinZ. Interestingly, T2S/H31K nisinZ was more stable than nisinZ at higher temperatures and neutral or alkaline pH.3. Little is reported about the effect of the amino acid of B ring on structure-function of nisinZ. We constructed mutant T8S nisinZ leding to a lanthionine residue in ring B on nisinZ molecule, instead of the original 3 -methyllanthionine, The result showed T8S nisinZ had the slightly less antimicrobial activity than nisinZ. the spectrum of antimicrobial activities and the solubilities were similar to nisinZ. However, T8S nisinZ was more stable than nisinZ at higher temperatures and neutral or alkaline pH.4. At present, the hinge region of nisinZ is rarely investigated, it has been revealed that the hinge region flexibility played a critical role in pore formation activity and the biological activity so that the research of the hinge region has been the hot point. However, few systematic studies of hinge region of nisinZ concerning its relationship of function-structure and its properties have been reported. In order to elaborate the role ofhinge region for biological activity and functional properties of nisin, we selected sixteen nisinZ mutants in the respective hinge region with the aim to study the role of charge, different mass amino acid and hydrophobic residue in hinge region on the relation of structure and function, and compared their activities and functional properties such as antimicrobiol spectrual and chemical stability and solubility. The results showed K22E nisinZ introducing of a negatively charged Glu residue in position 22 led to the loss production. N20E and M21E mutants had a large decrease in production and almost lost their activity.The antimicrobial activities of nisinZ variants in hinge region were determined as minimal inhibitory concentration(MIC) value against two indicator strains M. flavus and 5". thermophilus, N20H > N20Q , M21G , M21H , K22G , K22H , N20K/M21Kand N20F/M21L/K22Q had slightly less antimicrobial activities for M. flavus, their MIC values were 1-2 fold higher as compared to wild-type nisinZ, for S. thermophilus, their MIC values were 3~8 higher. Whereas N20K and M21KnisinZ showed a biological activity very similar to that of nisinZ.The spectrum of inhibitory activities: N20K and M21K nisinZ displayed antimicrob...
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