| Lactic acid bacteria (LAB) are no dout the most widely used probiotics with food grade status.Though China abounds in the resources of LAB, their research is still at the primary stage,expecially on bacteriocins produced by them, such as Nisin. Fueled up by the basic researches andcommercially useages of LAB, the construction of new industrial strains with excellent properties,specifically employing the advanced biotechnology (such as Protein Engineering, GeneEngineering and Cell Engineering et al.), not only confers food scientists' good opportunity aswell as big challenge, but also will fundamentally change the traditional applying of LAB inChina.A reliable expression system, like worldwide popular NICE (Nisin-Controlled geneExpression) using the Nisin promoter, is essencial in trying to enhance the beneficial functions ofLAB when employing them to express certain homo- or heterologous proteins. Researches relatedto promoter serve for the following construction of gene expression system, and also lead usnearer to the understanding of the prokaryotic expression and regulation process, becausepromoter, as the first necessary element that controls initiation of gene transcription, plays themost important role in an expression system.LABs were first isolated from traditional dairy products through basic morphologicalobservation. Then the isolates resisted to certain amount of Nisin were selected using twofolddilution method and some of them were identified by the 16S rRNA sequencing method.These Nisin-resisted stains were screened about their inhibiting substances production usingMicrococcus flavus as an indicator. And comparative studies on the physico-chemical properties(stability when treated by heat, pH and enzymes) were conducted between the inhibitingsubstances and Nisin. The Nisin-producing strain was determined by combining the two results ofthe 16S rRNA characterization and the comparative Studies.The nisA promoter fragment was amplified from Nisin-producing strain by PCR and insertedinto promoter-probe vector pNZ273, followed by histochemical experiment to check theβ-glucuronidase activity, aiming at verifying the function of the cloned fragment.The results were as follows:First, 90 strains of LAB were morphologically selected from traditional dairy productscollected from Inner Mongolia, of which, 50 strains were coccobacteria.Second, 15 out of 50 strains grew well on MRS agar when the Nisin conceration was 0.3×10-3g·mL-1. Most of the Nisin-resisted strains did not grow at the Nisin conceration of 1.25×10-3g·mL-1, as easily seen from the twofold dilution method detection experiment. Third, 4 out of the 15 Nisin-resisted strains produced some kind of antimicrobial substancesinhibiting the growth of Micrococcus flavus. And the inhibiting substance produced by a straindesignated S7 was stable when treated by heat (121℃, 15 min). The substance was more stablein acid environment (pH=2) than in neutralized condition (pH=7). Andα-chymotrypsin candegrade the substance. All of these charateres were the same as Nisin.Fourth, the S7 strains mentioned above were identified to be Lactococcus.lactis by 16S rRNAsequencing.Fifth, a 214 bp fragment exactly the same to the published Nisin promoter sequence wasamplified from S7 by PCR.Sixth, a recombined plasmid pDU273 was constructed by inserting the cloned fragment intopromoter-probe vector pNZ273.Seventh, after transformed pDU273 into L. lactis NZ9000, the gusA gene was expressedwhen induced by Nisin at the conceration of 20 ng·mL-1.From this study, it was concluded:First, the twofold dilution method was feasible in selecting Nisin-resisted strains by graduallyincreasing the Nisin conceration.Second, the 16S rRNA sequence identification has become a conventional technology inbacteria identification. And with the explosive increasing of 16S rRNA sequences submitted to theGenBank, this technology will be more and more prevalent with its fast and precise character.Third, it was verified that the cloned fragment could induce expression of the reporter gusAgene in pNZ273, indicating suitable usege as a nisA promoter.
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