Dissertation
Dissertation > Biological Sciences > Microbiology > Microbial biochemistry

Purification and Partial Characterisation of Enterocin SAU-2

Author ZhouJia
Tutor LiuShuLiang
School Sichuan Agricultural University
Course Of Food Science
Keywords bacteriocin Enterococcus purification characterisation structural gene
CLC Q936
Type Master's thesis
Year 2011
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Bacteriocin of lactic acid bacteria (LAB) is a kind of antibacterial peptide or precursor peptide produced by LAB which is "generally recognized as safe (GRAS)". Because of its safety, efficiency, no residue, non-toxicity and no resistance, there are many researches being input. Up to now, there are more than one hundred bacteriocins of LAB been found, but only Nisin is widely used. Nisin shows antimicrobial activity just when it is in the acidic condition, which is badly restricted its application. So it is significant to exploit bacteriocins which are high efficient, broad-spectrum and stable.Thirty-four strains of suspected LAB which were spherical isolated from milk residue were tested for antimicrobial activity against Micrococcus luteus 10209 by the punch-hole method. After eliminating the interference of organic acid and hydrogen peroxide, only cell-free supernatant of SAU-2 showed antimicrobial activity, and it inactivated after treatment with trypsin and proteinase K. The extract by ammonium sulfate precipitation strongly inhibited the growth of indicator bacteria. It was confirmed that strain SAU-2 was a bacteriocin producer, whose colonial morphology, phenotype characteristics and 16S rDNA sequence analysis showed high homology to Enterococcus, so it was identified as Enterococcus. The hemolytic phenotype of Enterococcus sp. SAU-2 was positive, but agg、gelE、cylM、cylB、cylA、esp、efaAfm、cpd、cob、ccf、cyIL、cyILs、fsrB and hyLEfm genotype were negative. It indicated that Enterococcus sp. SAU-2 was safe. Bacteriocin produced by LAB isolated from milk residue was reported for the first time.Contrasting the results of crude extract by ammonium sulfate precipitation, adsorption and ultrafiltration,90% ammonium sulfate precipitation was finally chosen for concentrating bacteriocin. Enterocin SAU-2 was partially purified by 90% ammonium sulfate precipitation followed by SP Sepharose Fast Flow. Acticity was eluted by a stepwise elution(0.5%,1.5%, 3.0%NaCl in 0.02 M sodium acetate buffer). The purification resulted in a 10.79-fold increase in specific activity and a yield of 17.27%. The estimated molecular weight of enterocin SAU-2 was approximately 4.35 kDa based on Tricine-SDS-PAGE, in which bacteriocin activity was confirmed by overlayer techniques were in accordance with this value. Primers of several enterocin structural genes were synthesized to detect the structural genes of enterocin SAU-2, and Enterococcus sp. SAU-2 showed only enterocin L50A structural genes whose size was identical as expected. Structural genes and amino acid sequences of enterocin SAU-2 had high homology with those of enterocin MR10A. It indicated that enterocin SAU-2 was a new member of bacteriocins.Partially characterisation of culture supernatant and crude extract were studied. The bacteriocin showed rather good resistance to both acid and alkali. Culture supernatant was active at pH values between 2~9,2~10 to salting-out liquid, and 2~11 to crude enterocin SAU-2. And the bacteriocin was heat resistant. After culture supernatant, salting-out liquid and crude bacteriocin were treated at 121℃for 20 min, the remaining activities were respectively 92%,97%,96% of the original activity. The bacteriocin inactivated by trypsin and proteinase K, but remained activities after treatment with papain and pepsin. The bacteriocin demonstrated antimicrobial activity towards seven strains of lactic acid bacteria and eight strains of Gram-positive bacteria, but show no antimicrobial activity towards Gram-negative bacteria and fungus, except for one Pseudomonas aeruginosa and one Rhodotorula glutinis. It was identical to most of bacteriocins. Three kinds of extract had almost the same antibacterial spectrum, and antibacterial activities of salting-out liquid and crude bacteriocin were more efficient than culture supernatant. It indicated that activity enhanced following purification the bacteriocin. MIC(Minimal Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) of enterocin SAU-2 against Listeria monocytogenes GIM1.228 were 256 AU/mL and 512 AU/mL respectively. And it displayed bactericidal mode of action with high killing action.The results of this paper didn’t only provide fundamental basis for researching structure, mechanism of action, cloning and expression of enterocin SAU-2, but also provided valuable data for using it as food preservative. This paper showed theoretical significance of researching bacteriocins and potential application of it.

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