• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.619-623
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• 1991

To investigate nisin production and resistance of Lactococcus lactis ssp. tactis ML (L. lactis $ML_8$, effects of medium, pH of culture broth, and cell growth on the nisin activity, and effect of nisin with or without $Ca^[2+}$ ion on the growth of L. lactzs were analyzed. In the bio-assay of nisin by the agar diffusion method, inhibition-zone diameter of Micrococcus Javus was propotional to the logarithm of nisin concentration ranged 0.5~20 unitlml (12.5~500 ng/mf). Nisin activity of the pasteurized culture filtrates of L. lactis MLs was high at pH 2!3 but was inactivated completely at pH over 6.0. Nisin production of the L. lactis $ML_8$ cultured on LTB broth increased at late logarithmic phase and reached 10.5 unitlml after 16 hr. The cell growth of L. lactis LM 0230, a plasmid free and nisin sensitive strain, was inhibited on agar medium containing 7 unitlrnl of nisin, while L. lactis $ML_8$ showed high survival ability at 20 unitld of nisin. When 40 mM $Ca^[2+}$ ion was added to Elliker broth with 8 unitlml of nisin, the growth pattern of L. lactis $ML_8$ was similiar to that on control medium which did not contain nisin and $Ca^[2+}$ ion, and this suggested that $Ca^[2+}$ increased the nisin resistance of the L. lactis.

• 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.

• Preventive Nutrition and Food Science
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• v.5 no.3
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• pp.153-159
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• 2000

A 4.4 kb DNA fragment encompassing lacA (galactoside acetyltransferase) and lacZ($\beta$-galactosidase) genes from Lactococus lactis ssp. lactis ATCC 7962 (L. lactis 7962) was introduced ito a Lac strain, Lactococcus lactis ssp. lactis MG1363 (L. lactis MG1363) by using a lactococcal expression vector, pMG36e and expression level of lacZ was examined. Growth rates and $\beta$-galactosidase ($\beta$-gal) activities of MG1363 cells carrying recombinant plasmid, pMLZ3, on M17 broth containing different carbon sources (1%, w/v) were examined. Contrary to the expectations, MG1363 [pMLZ3] grown on lactose showed the lowest enzyme activity (17 units) and cells grown on galactose had the highest $\beta$-gal activity (41 units). Cells grown on glucose had intermediate activity (33 units). These activities are about one tenth of the values observed in L. lactis 7962 where lacZ is present as a single-copy gene in the chromosome. When the cellular concentrations of lacZ transcript were examined using slot blot hybridization, it was found that MG1363[pMLZ3] produced sufficient amounts of transcript. These results indicate that either proteolytic degradation of $\beta$-gal or other regulatory mechanism prevent the translation or accumulation of $\beta$-gal in L. lactis MG1363 cells. In regard to regulation, the presence of the ccpA gene in L. lactis MG1363 was confirmed by Southern blot.

• 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.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.281-284
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• 1991

To investigate the optimum conditions for the production of ammonia as a precursor of tetramethylpyrazine flavor compound from arginine by Lactococcus lactis ssp. lactis biovar. diacetilactis FC1, fermentation factors such as initial pH of culture media, fermentation temperature, concentration of arginine-HC1, and sugars were examined. The optimum conditions were initial pH 5.5 of the culture media, fermentation temperature of $34^{\circ}C$, 6% (w/v) of arginine-HC1, and 1% (w/v) of galactose as a carbon source. Under the optimum fermentation conditions, 40 mmole/l of ammonia was produced after 40 h.

• Journal of Microbiology and Biotechnology
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• v.13 no.1
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• pp.134-138
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• 2003

The secondary and tertiary structures of ${\beta}$-galactosidase from L. lactis ssp. lactis 7962 were designed using Nnpredict and Sybyl version 6.3. By using site-directed mutagenesis, the mutated enzymes, Tyr-475-phe and Glu-506-Asp, were generated based on the structural modeling of L. lactis ssp. lactis 7962. The enzymes Tyr.-475-Phe and Glu-506-Asp had <$1\%$ of the activity of the native enzyme with ONPG as substrate. The $V_{max}$ values of the mutated enzymes were greatly reduced (1,800~40,000-1314) compared with the value for the native ${\beta}$-galactosidase. However, the $K_m$ values of Tyr-475-Phe and Glu-506-Asp with ONPG, PNPG, PNPF, and PNPA were not significantly different from those of the native enzyme. The results obtained support the suggestion that Tyr-475 and Glu-506 constitute very important parts of the catalytic machinery of the ${\beta}$-galactosidase.

• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.393-397
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• 1999

The gene (galE) encoding UDP-galactose-4-epimerase, operative in the galactose metabolic pathway, was cloned together with the $\beta$-galactosidase gene (lacZ) from Lactococcus lactis ssp. lactis ATCC7962 (L. lactis 7962). galE was found to have a length of 981 bps and encoded a protein with a molecular mass of 36,209 Da. The deduced amino acid sequence showed a homology with GalE proteins from several other microorganisms. A Northern analysis demonstrated that galE was constitutively expressed by its own promoter. When galactose or lactose was added into medium, the galE transcription was induced by several upstream promoters. The structure of the gal/lac operon of L. lactis 7962 was partially characterized and the gene order around galE was galT-lacA-lacZ-galE-orfX.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.201-205
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• 1999

The effects of growth temperature and a carbon source on the expression of $\beta$-galactosidase gene of Lactococcus lactis ssp. lactis ATCC 7962 (L. lactis 7962) were investigated. At $25^{\circ}C$, L. lactis 7962 had a higher $\beta$-galactosidase activity than cells grown at $30^{\circ}C$ or $37^{\circ}C$, although cells grew most quickly at $37^{\circ}C$ The highest $\beta$-galactosidase activity was observed in cells grown in M17 with lactose (l %) followed by cells grown in a galactose (1 %) medium. L. lactis 7962 exhibited the minimum $\beta$-galactosidase activity in glucose media, indicating catabolite repression. When the cellular levels of $\beta$-galactosidase mRNA were examined using slot blot hybridization, no significant differences were observed between cells grown at $25^{\circ}C$ and cells at $30^{\circ}C$ or $37^{\circ}C$ in the same media. This suggests that the quantity of $\beta$-galactosidase mRNA may not be the reason for the higher $\beta$-galactosidase activities of L. lactis 7962 at $25^{\circ}C$ The level of ccpA (Catabolite Control Protein) transcript remained almost constant during the exponential growth phase irrespective of a carbon sourse.

• Journal of Microbiology and Biotechnology
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• v.16 no.5
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• pp.786-790
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• 2006

Cellular components of Lactococcus lactis ssp. lactis (heat-killed whole cells, cytoplasm, and cell walls) were tested for their in vivo immunopotentiating activity. Peritoneal macrophages from mice orally administered with heat-killed whole cells exhibited significantly greater phagocytic activity than the groups administered with cell-wall fraction or cytoplasm fraction. The cytotoxicity of natural-killer cells was the highest in the group administered with whole cells, and the production of cytokines ($IFN-\gamma$, IL-2, and IL-12) in spleen cells was significantly higher, when cellular components were injected, and it tended to be higher in the cell-wall and cytoplasm groups than in the whole-cell group. Interestingly, the cytokine production of Peyer's patch cells was high, when cytoplasm fractions were administered. These results demonstrate that whole cells and cytoplasm and cell-wall fractions of L. lactis ssp. lactis have immunopotentiating activities, which are related to the stimulation of Peyer's patches.

• 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.