| Summary: | 碩士 === 國立中興大學 === 食品科學系 === 92 === The antimicrobial peptide nisin belongs to the family of lantibiotics and is produced by several strains of Lactococcus lactis. Lactic acid bacteria (LAB) have been used for centuries in the preparation and processing of foods, beverages and animal feed due to its food-grade status. In this study, We aimed to develop a food-grade expression system in lactic acid bacteriaby taking advantage of the nisin immunity (nisI) and nisin resistance (nsr) gene from L. lactis BCRC10791, BCRC14016 and pFM011, respectively instead of using the common antibiotic resistance genes as selection markers.
In previous study, we had chemically synthesized a promoter derived from B. subtilis veg promoter. A hagp Up element consensus -35 (TTGACA) and -10 (TATAAT) hexamers, and a TG motif were designed to construct the expression signals. In order to analysis the function of the expression signals, gusA was used as a reporter gene. Functional analysis of expression signals showed that the fusion of endogenous gusA to an artificial σA-type promoter constitutively expressed in Lactococcus lactis NZ9000; however, lower GusA activities was found for Lactobacillus paracasei BCRC14023. The secretable reporter levanase (sacC) controlled by the artificial σA-type promoter is able to be expressed extracellularly in L. lactis NZ9000 and Lb. paracasei BCRC14023. To develop a new selection system for lactic acid bacteria, the food grade selection markers, nisI and nsr were amplified from L.lactis BCRC10791 as well as BCRC14016 and from pFM011 by PCR. The amplified product were then cloned into pET29a and pHAσAGus and transformed into B.subtilis DB104. Both nisI and nsr gene elevated the nisin resistance level in Bacillus subtilis DB104. From the nisin tolerance tests of L. lactis NZ9000 and Lb. paracasei BCRC14023, 50 IU and 500 IU nisin concentrations was chosen for food grade vecter selection in L. lactis NZ9000 and Lb. paracasei BCRC14023, respectively. Food grade vector with nsr gene enhances the nisin tolerance of L. lactis NZ9000 and Lb. paracasei BCRC14023 efficiently. Food grade vector with nisI gene are not so effective, maybe due to the low expression level.
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