Aminopeptidase A (Pep A) is a metal-dependent enzyme that specifically hydrolyze peptides with the N-terminal amino acids glutamic acid (Glu) and aspartic acid (Asp). A possible application of PepA is the hydrolysis of Glu/Asp-rich food proteins such as wheat gluten and casein, increasing the flavor and solubility of food protein. In the present study, the gene encoding a Pep A from Lactococcus lactis ssp. lactis IL1403 was synthesized and introduced into Pichia pastoris GS115 (His4). Lc-Pep A was successfully expressed and secreted to the culture medium, followed by identification and purification to homogeneity. Characteristics study demonstrated that Lc-Pep A could specifically hydrolyze the substrates Glu-pNA and Asp-pNA with similar catalytic activity, and this was further confirmed by the kinetics parameters measured. Additionally, Lc-Pep A showed a broad thermostability and pH stability with an optimum temperature of 60 ℃ and an optimum pH of 8.0. The enzyme activity of Lc-Pep A was activated by metal ions Co²⁺, Mn²⁺, and Zn²⁺ but was strongly inhibited by Ni²⁺and Cu²⁺. The routine proteinase inhibitor had no effect on the activity of Lc-Pep A. However, Lc-Pep A was strongly inhibited by the metallopeptidase inhibitor, EDTA, and disulfide bond-reducing agents. The study may facilitate production and application of Lc-Pep A.
Glutamyl Aminopeptidase ; Lactococcus lactis/genetics* ; Biological Transport ; Culture Media ; Glutamic Acid

Aims: Lactococcus lactis is a non-colonizing, generally-regarded as safe (GRAS) lactic acid bacteria that has been frequently studied as a potential vector for bactofection. To mediate bactofection, a series of interaction between the bacteria and the host cell needs to occur. This study aims to investigate the in vitro bacterial-cell interaction between a locally-isolated L. lactis M4 strain with human colorectal cancer line, Caco-2. Methodology and results: Bacterial interaction was evaluated via adherence and internalisation assays. A 250:1 ratio of bacteria to cancer cell was selected as the optimum multiplicity of infection for all assays. After 2 h, L. lactis M4 was able to adhere to and internalise into Caco-2 cells at comparable rates to commercial strains L. lactis NZ9000 and MG1363. Conclusion, significance and impact of study Findings from this study showed that this strain has similar interaction properties with the commercial strains and would make a promising candidate for future bactofection studies and development of bacteria-mediated DNA vaccination against various diseases.
Lactococcus lactis ; Colorectal Neoplasms ; Caco-2 Cells

Lactococcus lactis is a gram-positive cocci used extensively in the dairy industry, but considered an unusual pathogen in humans. Among its five subspecies, L. lactis subsp. lactis in particular has rarely been reported as a pathogen. We report a case of septic shock caused by L. lactis subsp. lactis in an adult patient. A 64-yr-old male patient was admitted to outpatient clinics, with chief complaints of fever and chills for one week after convalescent hospital admission. He had severe ileus requiring surgery. He had a peripherally inserted central catheter from convalescent hospital, which was immediately removed. From two sets of blood and catheter tip cultures, we identified L. lactis subsp. lactis using the Vitek 2 system (bioMerieux Inc., USA), and confirmed this result by 16S rRNA sequencing. The patient was empirically treated with ciprofloxacin, and he recovered and was discharged.
Adult* ; Ambulatory Care Facilities ; Catheter-Related Infections* ; Catheters ; Chills ; Ciprofloxacin ; Fever ; Gram-Positive Cocci ; Hospitals, Convalescent ; Humans ; Ileus ; Lactococcus lactis* ; Lactococcus* ; Male ; Shock, Septic*

Aims: This study aimed to investigate the suitability and efficacy of various encapsulation media in bioencapsulating the probiotic Lactococcus lactis subsp. lactis in Artemia franciscana nauplii. The impact of the encapsulation media on nauplii survival and probiotic recovery was also determined. Methodology and results: Various encapsulation media (sodium alginate, palm oil, starch, gum Arabic and gelatin) were prepared by dissolving the respective media in artificial sea water. Each media was prepared in four different concentrations: 0.25, 0.5, 1.0 and 2.0 g/L. To determine the suitability of encapsulation media on the survivability of A. franciscana, survival rate (SR) of Artemia nauplii was determined after 8 hours post-encapsulation. Instar II stage Artemia nauplii at 1 nauplii per mL was used for each replicate. The result revealed that A. franciscana reached 100 % SR in the encapsulation media at ≤ 0.5 g/L. All media enabled > 23 % recovery of L. lactis subsp. lactis from encapsulated A. franciscana, which is similar (p > 0.05) to the recovery of free-cells (non-encapsulated) of L. lactis subsp. lactis. Noticeably in sodium alginate (E1) treatment, the total counts of L. lactis subsp. lactis in bioencapsulated A. franciscana were the highest among others, accounting for 2.44 × 107 CFU/mL per A. franciscana tissue homogenate. Conclusion, significance and impact of study: Artemia nauplii bioencapsulated with L. lactis subsp. lactis using 0.5 g/L sodium alginate as encapsulation medium has the highest SR for nauplii and bioencapsulation efficiency, respectively. This result provides a basic guideline for Artemia bioencapsulation in fish/shrimp larval culture.
Lactococcus lactis

To obtain active protein of pIL-18 expression in Lactococcus lactis, and to observe its biological activity, the total RNA was extracted as template from peripheral blood mononuclear cells. Porcine interleukin 18 (pIL-18) was amplified by RT-PCR. The resulting fragment was cloned into pAMJ399 L. lactis vector, and then transformed to L. lactis MG1363 cells by electroporation. Expression of pIL-18 protein was detected by SDS-PAGE and Western-blotting. Bioactivity of the product was tested by pig spleen lymphocyte proliferation test and cytopathogenic effect inhibition assay. The result of Western blotting and bioactivity test shows that the molecular weight of pIL-18 protein was 19 kDa. The react line was observed in both supernatant and precipitated of the recombinant bacteria pAMJ399-pIL18/MG1363. The expressed pIL-18 can promote the proliferation of pig spleen lymphocyte, and significantly inhibit virus multiplication. As conclusion, porcine interleukin-18 was successfully expressed in L. lactis, and the product was biologically active.
Animals ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Electroporation ; Genetic Vectors ; Interleukin-18 ; biosynthesis ; Lactococcus lactis ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Swine

Lactococcus lactis (L. lactis) is an important gram-positive bacterium in dairy products. It is a rare cause of opportunistic infections with only four cases of Lactococcus peritoneal dialysis (PD) peritonitis reported in the literature. In Korea, L. lactis infection was first reported in a liver abscess patient in 2010; however, PD peritonitis with Lactococcus has not been reported in Korea. Recently, we experienced a case of Lactococcus-associated polymicrobial PD peritonitis. The patient was initially managed with broad-coverage antibiotics; however, owing to a poor response, the PD catheter was removed and the patient was switched to hemodialysis. We discuss this case and review the literature.
Anti-Bacterial Agents ; Catheters ; Dairy Products ; Humans ; Korea ; Lactococcus ; Lactococcus lactis ; Liver Abscess ; Opportunistic Infections ; Peritoneal Dialysis ; Peritonitis ; Renal Dialysis

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.
Anaerobiosis ; Escherichia coli ; enzymology ; genetics ; metabolism ; Fermentation ; Genetic Engineering ; Glucose ; metabolism ; Industrial Microbiology ; Lactococcus lactis ; enzymology ; NAD ; metabolism ; Pentosyltransferases ; biosynthesis ; genetics ; Pyruvate Carboxylase ; biosynthesis ; genetics ; Succinic Acid ; metabolism

Nisin is an antimicrobial peptide widely used in food industry. In this study, Nisin A production in Lactococcus lactis ATCC 11454 was improved by overexpression of Nisin A structural gene nisA through introducing a shuttle expression vector pMG36ek-nisA and an integrated vector pDG780-nisA into the host strain. The differences of growth profiles and Nisin A production level between the two obtained genetic engineering strains FMM1/FMM2 and the parent strain were investigated. Our results show that while the growth profile (the growth rate, biomass and pH) of FMM1 was similar to the parent strain, its Nisin A production increased 31%. In contrast, the biomass of FMM2 was notably lower than the parent strain, while its yield of Nisin A enhanced slightly. The transcription level of genes involved in Nisin A biosynthesis in both engineering strains was further detected by RT-PCR. We found that all the 11 Nisin A biosynthetic genes in FMM1 and FMM2 had a higher transcription level than those in the parent strain, and these genes exhibited more significant increasing degree of transcription level in FMM1 which hosted the autonomous replicating nisA gene. These data suggest that expression of nisA may act as a rate-limit factor in Nisin A biosynthesis. In conclusion, this work provides a new method to improve Nisin A production by increasing the transcription level of nisA, paving the way to further large-scale industrial production of Nisin A.
Bacterial Proteins ; genetics ; Genes, Bacterial ; Genetic Engineering ; Genetic Vectors ; Lactococcus lactis ; genetics ; metabolism ; Nisin ; biosynthesis ; genetics ; Transcription, Genetic

The possibility of heterologous expression of human Stearoyl-CoA Desaturase (scd1) was investigated. The scd1 encoding sequence was inserted into the pNZ8149 to generate the pNZ8149-scd1 expression plasmids. Then we introduced the pNZ8149-scd1 construct into the Lactococcus lactis NZ3900 to investigate its enzyme activity. The results show that heterologous expressed SCD1 enzyme resulted in a 92%-169% increase in the C16:1n-7 and a 53-127% increase in the C18:1n-7 (P<0.05). The SCD1 enzyme was capable of producing n-7 fatty acids in Lactococcus lactis efficiently. It also suggests that the fatty acid desaturases can be heterologous expressed in Lactococcus lactis to produce the helpful fatty acids.
Electroporation ; Humans ; Lactococcus lactis ; genetics ; metabolism ; Mutagenesis, Insertional ; Nisin ; pharmacology ; Recombinant Proteins ; biosynthesis ; genetics ; Stearoyl-CoA Desaturase ; biosynthesis ; genetics

Lactococcus lactis cremoris infections are very rare in humans. We experienced liver abscess and empyema due to L. lactis cremoris in an immunocompetent adult. A 42-yr-old man was admitted with fever and abdominal pain. Abdominal computed tomography (CT) revealed a liver abscess and chest CT showed loculated pleural effusion consistent with empyema. L. lactis cremoris was isolated from culture of the abscess material and blood. The patient was treated with pus drainage from liver abscess, video-assisted thoracoscopic decortications for empyema, and antibiotics including cefotaxime and levofloxacin. The patient was completely recovered with the treatment. To our knowledge, this is the first report of a L. lactis cremoris infection in Korea.
Adult ; Anti-Bacterial Agents/therapeutic use ; Cefotaxime/therapeutic use ; Drainage ; Empyema/*diagnosis/*microbiology/surgery ; Gram-Positive Bacterial Infections/complications/*diagnosis/drug therapy ; Humans ; *Lactococcus lactis/drug effects/isolation & purification ; Liver Abscess/*diagnosis/*microbiology ; Male ; Microbial Sensitivity Tests ; Ofloxacin/therapeutic use ; Thoracic Surgery, Video-Assisted ; Tomography, X-Ray Computed