• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.241-247
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• 2005

A new lactococcal shuttle/expression vector for lactococci, pWgal13T, was constructed using a $\beta$-galactosi-dase gene (lacZ) from Lacfococcus lactis ssp. lactis ATCC 7962 as a screening marker. The pWgal 13T was introduced into Escherichia coli DH5a and L. lactis MG1363, and was easily detected by the formation of blue colonies on a medium containing X-gal without any false transformants. Also, the quantitatively lacZ activity of pWgal13T was measured in L. lactis ssp. cremoris MG1363, and was found to be four times higher than that of L. lactis ssp. lactis ATCC7962 grown on a medium containing glucose, which shows that the lacZ gene of pWgal13T can be used for the efficient screening of L. lactis on general media. The pWgal13T was equipped with a lactococcal replicon of pWV01 from L. lactis Wg2, the new promoter P13C from L. lactis ssp. cremoris LM0230, multiple cloning sites, and a terminator for the expression of a relevant gene. The vee-tor pWgal13T was used for the expression of the EGFP gene in E. coli and L. lactis. These results show that the lactococcal expression/shuttle vector constructed in the present study can be used for the production of foreign proteins in E. coli and L. lactis.

• Journal of Microbiology and Biotechnology
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• v.6 no.2
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• pp.137-141
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• 1996

Acetoin and ammonia, the precursors of tetramethylpyrazine (TMP) having "meaty" or "roasted" flavors, were produced by the culture of Lactococcus lactis ssp. lactis biovar. diacetilactis FC1 in free and immobilized cell systems. Cells were immobilized using k-carrageenan and then were incubated at $34^{\circ}C$. The TMP productivity (0.34 g/l) and the conversion ratio (9.3%) of acetoin to TMP of the immobilized cell system were higher than those (0.24 g/l, 7.0%) of the free cell system. When the beads were activated for 12 h, the productivity of acetoin and TMP increased slightly.

• Journal of Microbiology and Biotechnology
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• v.1 no.3
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• pp.202-206
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• 1991

To produce acetoin as a precursor of the tetramethylpyrazine flavor compound from citrate by Lactococcus lactis ssp. lactis biovar. diacetilactis FC1, fermentation factors such as inital pH of culture media, temperature, concentration of Na-citrate, thiamin-HC1 and sugars were examined. The best acetoin production was achieved with initial pH in the culture media of 5.5, fermentation temperature of $34^{\circ}C$, Na-citrate concentration of 3%, addition of thiamin-HC1 at 2 mg/l and galactose as a carbon source. When fermentation was carried out under the optimum conditions, the exhaustion of Na-citrate and the production of acetoin took simultaneously and acetoin reached the maximum content, 80 mmole/l after 20 hours.

• Journal of Microbiology and Biotechnology
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• v.17 no.9
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• pp.1491-1497
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• 2007

Arginine deiminase (ADI, E.C. 3.5.3.6), one of the arginine deprivation enzymes, exhibits anticarcinogenic activities. The present study investigated the anti-inflammatory activities of the purified recombinant ADI originating from Lactococcus lactis ssp. lactis ATCC7962 (LADI). LADI dose-dependently inhibited lipopolysaccharide (LPS)-induced upregulation of inducible nitric oxide synthase and the production of nitric oxide in RAW 264.7 murine macrophages. The induction of cyclooxygenase-2 expression and subsequent production of prostaglandin $E_2$ by LPS was also attenuated by LADI treatment. Moreover, LADI inhibited the production of interleukin-6 in LPS-stimulated RAW 264.7 macrophages. These results indicate that LADI exerts anti-inflammatory effects, which may in part explain its chemopreventive potential.

• KSBB Journal
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• v.11 no.4
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• pp.504-509
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• 1996

Promoters which are useful for constructing expression vectors for lactic acid bacteria were obtained from the chromosomal DNA of Lactococcus lactis ssp. lactis MG1363. pBV5030, a promoter-selection vector, replicates in L. lactis and Escherichia coli and carries a promoterless chloramphenicol acetyltransferase gene (cat-86). After examining E. coli transformants which grew on LB media containing chloramphenicol (Cm, 20$\mu\textrm{g}$/mL) , many MG1363 derived DNA fragments which encompass promoter sequences were identified. Some recombinant E. coli cells can grow at the Cm concentration of 1,000$\mu\textrm{g}$/mL. When plasmids from those highly resistant E. coli cells were purified and introduced into L. lactis ssp. lactis MG1614 cells by electroporation, lactococcal transformants showing Cm resistance were obtained. So far, five plasmids with different promoter inserts were introduced into L. lactis MGl614 cells. The maximum level of Cm resistance in L. lactis MG1614 transformants was quite low (20$\mu\textrm{g}$/mL) when compared with that observed in recombinant E. coli cells harboring the same plasmids.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.154-154
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• 2001

• Microbiology and Biotechnology Letters
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• v.27 no.3
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• pp.260-265
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• 1999

The structural $\beta$-galactosidase gene (lacZ) from Lactococcus lactis ssp. lactis 7962 was cloned into plamid vector pKF18, which was designated as pKF-gal. Expression of the lacZ from L. lactis 7962 was found to be higher when cells were grown at 3$0^{\circ}C$ than 37$^{\circ}C$. Maximum $\beta$-galactosidase activity was obtained when E. coli/pKF-gal was cultivated for 6hr at 3$0^{\circ}C$ and for 3hr at 37$^{\circ}C$, and L. lactis 7962 was grown for 8hr at 3$0^{\circ}C$. Enzyme induction was achieved by the addition of lactose, galactose, or lactose+IPTG to growing culture. The addition of glucose had no effect on enzyme induction.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.285-287
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• 1991

To produce the tetramethylpyrazine (TMP) flavor compound, Lactococcuss lactis subsp. lactis biovar. diacetilactis (L. diacetilactis) FC1 was cultivated in the TMP medium containing 3% (w/v) of Na-citrate and 6% (w/v) arginine-HC1 as substrates of acetoin and $NH_3$, respectively, which are the two precursors of the TMP. After 19-day fermentation at $34^{\circ}C$, 0.57 g/l or 4.19 mmole/l of the TMP was produced. This was the first result showing that the TMP could be produced by L. diacetilactis.

• Journal of Microbiology and Biotechnology
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• v.2 no.2
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• pp.73-77
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• 1992

The effect of a nisin-producing Lactococcus lactis spp. lactis (L. lactis) on the growth and attachment of Listeria monocytogenes Scott A and Brie 1 on stainless steel and their growth in Brain Heart Infusion broth was determined. Viable cells of Listeria decreased rapidly after 9~12 hr of incubation at $21^{\circ}C$ and after 6~9 hr of incubation at $32^{\circ}C$ in the presence of L. lactis. The number of L. monocytogenes Scott A attached to stainless steel in pure culture was $2.5{\times}10^3/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}2.3{\times}10^3/\textrm{cm}^2{\;}at{\;}32^{\circ}C$ after 48 hr of incubation, but was only $10/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}1.1{\times}10/\textrm{cm}^2{\;}at{\;}32^{\circ}C$ in the presence of L. lactis. Results from L. monocytogenes strain Brie 1 were similar to those from strain Scott A. The population of L. monocytogenes Scott A which attached to stainless steel with previously adherent L. lactis was $1.8{\times}10^2/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}8.2{\times}10^2/\textrm{cm}^2{\;}at{\;}32^{\circ}C$, whereas the population attached to sterile stainless steel was $1.2{\times}10^3/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}2.1{\times}10^2/\textrm{cm}^2{\;}at{\;}32^{\circ}C$. For L. monocytogenes Brie 1, the attached population of the control was $1.6{\times}10^4/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}3.2{\times}10^2/\textrm{cm}^2{\;}at{\;}32^{\circ}C$, and on stainless steel with adherent L. lactis, it was $1.1{\times}10/\textrm{cm}^2{\;}at{\;}21^{\circ}C{\;}and{\;}6.9{\times}10/\textrm{cm}^2{\;}at{\;}32^{\circ}C$. Surface adherent L. lactis was less inhibitory to attachment of L. monocytogenes on stainless steel than a liquid culture inoculum. Listeria attached to stainless steel survived dry storage for 20 days both in the presence and absence of adherent lactococci.

• Journal of Microbiology and Biotechnology
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• v.3 no.4
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• pp.217-223
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• 1993

To investigate the nature and location of the nisin-resistance determinant of Lactococcus lactis subsp. lactis 7962 (L. lactis 7962), a total plasmid DNA prepared from L. lactis 7962, a nisin producer, was used to transform L. lactis subsp. lactis LM0230, a plasmid-free and nisin-sensitive strain, by protoplast mediated transformation procedures. All of the nisin-resistant transformants acquired the ability to utilize sucrose at the same time, confirming the close linkage between these two determinants in L. lactis 7962. The plasmid DNA profiles of a few selected nisin-resistant transformants were examined by agarose gel electrophoresis. No common plasmid was found among the transformants and some small plasmids previously not present in L. lactis 7962 were detected. These transformants were named as L. lactis KL1, KL2, KL3, KL4, or KL5, respectively based on their plasmid profiles. Growth curves of all transformants were similar to that of L. lactis LM0230, but different from that of L. lactis 7962. L. lactis KL5 showed the highest level of resistance to nisin, growing up to 1, 200 IU nisin/ml after 40 hr incubation. Some nisin-sensitive derivatives of KL1 or KL2 were obtained by plasmid curing experiments. The plasmid DNA profiles of the nisin-sensitive KL1 derivatives were apparently the same as that of the KL1. All of the nisin-sensitive KL2 derivatives were plasmid-free, but a nisin-resistant strain with no apparent plasmid was also obtained. These results indicate that the nisin-resistance of the $Nis^r$ transformants is presumably mediated by the chromosomally located gene(s) rather than plasmid-encoded gene(s).