Antimicrobial activity and cytotoxicity trait of a bioactive peptide purified from Lactococcus garvieae subsp. bovis BSN307T.

A bioactive peptide of 8595 Da was purified from the cell free supernatant of Lactococcus garvieae subsp. bovis BSN307T . MALDI MS/MS peptide mapping and the data base search displayed no significant similarity to any reported antimicrobial peptide of LAB. This peptide at a dose concentration of 200 µg ml-1 inhibited the growth of both Gram-positive and Gram-negative bacteria by 58-89% and a dose of 500 µg ml-1 scavenged 50% of DPPH-free radicals generated. Interestingly, cytotoxicity assay demonstrated that 17 µg ml-1 of peptide selectively inhibited 50% proliferation of mammalian cancer cell lines HeLa and MCF-7 whereas normal H9c2 cells remained unaffected. Fluorescent microscopic analysis after DAPI nuclear staining of HeLa cells showed characteristics of apoptosis and activation of caspase-3 was ascertained by caspase-3 fluorescence assay.

Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv.

AIM: Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical beta-lactamases activity. METHODS AND RESULTS: Analysis of the M. tuberculosis H37Rv genome revealed that Rv2068c, Rv0406c and Rv3677c gene products were predicted to exhibit beta-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have beta-lactamase activity by the hydrolysis of nitrocefin and other beta-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of beta-lactam antibiotics. CONCLUSION: The study revealed the possibility of more than one gene in M. tuberculosis, encoding proteins having beta-lactamase or beta-lactamase-like activity, giving wide spectrum of resistance against beta-lactams. SIGNIFICANCE AND IMPACT OF THE STUDY: Systematic study of hypothetical beta-lactamases of M. tuberculosis and related species and their correlation with beta-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.

Production of L-leucine aminopeptidase by selected Streptomyces isolates.

AIMS: To screen various Streptomyces cultures producing L-leucine aminopeptidase (LAP). METHODS AND RESULTS: Twenty-one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B-3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8.0-8.5 and the temperature optima varied between 50 and 65 degrees C. LAP of S. mobaraensis was stable at 60 degrees C and pH 8.5 for 60 min. Metal ion salts, CoCl(2).6H(2)O and ZnSO(4).7H(2)O in 0.7 mmol l(-1) concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa. CONCLUSIONS: Streptomyces mobaraensis NRRL B-3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N-terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.

Polyurethane foam as an inert carrier for the production of L(+)-lactic acid by Lactobacillus casei under solid-state fermentation.

AIM: Production of L-lactic acid in solid-state fermentation (SSF) using polyurethane foam (PUF) as inert support moistened with cassava bagasse starch hydrolysate. METHODS AND RESULTS: PUF impregnated with cassava bagasse starch hydrolysate as major carbon source was used for the production of L-lactic acid using Lactobacillus casei in solid-state condition. The key parameters such as reducing sugar, inoculum size and nutrient mixture were optimized by statistical approach using response surface methodology. More than 95% conversion of sugars to lactic acid from 4 g reducing sugar per gram dry support was attained after 72 h when the inert substrate was moistened with 6.5 ml of nutrient solution and inoculated with 1.5 x 10(9) CFU of L. casei. While considering the lactate yield based on the solid support used, a very high yield of 3.88 g lactic acid per gram PUF was achieved. CONCLUSION: PUF acted as an excellent inert support for L. casei and provided a platform for the utilization of starchy waste hydrolysate in a lower reactor volume. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a cost effective cultivation of lactic acid bacteria for producing lactic acid from agro based waste products such as cassava bagasse. This is the first report on the exploitation of PUF as an inert support for lactate production under SSF.

l-leucine aminopeptidase production by filamentous Aspergillus fungi.

AIMS: To screen various filamentous fungi belonging to Aspergillus spp. producing leucine and methionine aminopeptidases. METHODS AND RESULTS: Twenty-eight Aspergillus strains representing 14 species within the genus were screened for L-leucine aminopeptidase (LAP) production in two media in shake flask fermentation. Two Aspergillus sojae (NRRL 1988 and NRRL 6271) and one Aspergillus oryzae (NRRL 6270) strains were selected as the best producers for further studies. The peak LAP activities were 2.61, 2.59 and 1.30 IU ml(-1) for the three fungi on days 2, 5 and 4 respectively. In addition to LAP, L-methionine aminopeptidase (MAP) activity was also detected. Apart from submerged fermentation, the highest LAP yields by solid-state fermentation were 11.39, 17.40 and 13.02 IU g(-1) dry matter for the above fungi. The temperature and pH optimum of the enzyme was found to be in the range of 65-75 degrees C at pH 8.0-9.0 for all three fungi. Metal ions, Co(2+) and Fe(2+) in 2 mmol l(-1) concentration apparently enhanced the relative enzyme activity and heat stability. CONCLUSIONS: Two A. sojae (NRRL 1988 and NRRL 6271) and one A. oryzae (NRRL 6270) strains were found to be the best producers of LAP and MAP. The preliminary characterization studies revealed that the enzyme is considerably thermostable and belongs to the class metalloenzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of aspergilli were screened and the ability of the fungal aminopeptidase to release a particular N-terminal amino acid along with its high thermal stability, makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.

Expression of genes of lipid synthesis and altered lipid composition modulates L-glutamate efflux of Corynebacterium glutamicum.

L-Glutamate is made with Corynebacterium glutamicum on a scale of more than 106 tons/year. Nevertheless, formation of this amino acid is enigmatic and there is very limited molecular information available to unravel the apparently complex conditions leading to L-glutamate efflux. Here, we report the isolation and overexpression of the genes involved in lipid synthesis: acp, fadD 15, cma, cls, pgsA2, cdsA, gpsA, and plsC, and the inactivation of cma and cls. In addition, the consequences for phospholipid content, temperature sensitivity, as well as detergent-independent and detergent-dependent L-glutamate efflux were quantified. An in part strong alteration of the phospholipid composition was achieved; for instance, overexpression offadD15 encoding an acyl-CoA ligase resulted in an increase of phosphatidyl inositol from 12.6 to 30.2%. All strains, except that overexpressing acp (acyl carrier protein), exhibited increased temperature sensitivity, with the strongest sensitivity present upon cls (cardiolipin synthetase) inactivation. As a consequence of the genetically modified lipid synthesis, L-glutamate efflux changed quite dramatically; for instance, overexpression of plsC (acylglycerolacyl transferase) resulted in a detergent-triggered increase of L-glutamate accumulation from 92 mM to 108 mM, whereas acp overexpression reduced the accumulation to 24 mM. With some of the overexpressed genes, substantial L-glutamate excretion even without detergent addition was obtained when the fermentation temperature was elevated. These data show that the chemical and physical properties of the cytoplasmic membrane are altered and suggest that this is a necessary precondition to achieve L-glutamate efflux.

Biochemical characterization of acyl carrier protein (AcpM) and malonyl-CoA:AcpM transacylase (mtFabD), two major components of Mycobacterium tuberculosis fatty acid synthase II.

Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in the biosynthesis of fatty acids in all bacteria, including Mycobacterium tuberculosis. MCAT catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP, to generate malonyl-ACP, which is an elongation substrate in fatty acid biosynthesis. To clarify the roles of the mycobacterial acyl carrier protein (AcpM) and MCAT in fatty acid and mycolic acid biosynthesis, we have cloned, expressed, and purified acpM and mtfabD (malonyl-CoA:AcpM transacylase) from M. tuberculosis. According to the culture conditions used, AcpM was produced in Escherichia coli in two or three different forms: apo-AcpM, holo-AcpM, and palmitoylated-AcpM, as revealed by electrospray mass spectrometry. The mtfabD gene encoding a putative MCAT was used to complement a thermosensitive E. coli fabD mutant. Expression and purification of mtFabD resulted in an active enzyme displaying strong MCAT activity in vitro. Enzymatic studies using different ACP substrates established that holo-AcpM constitutes the preferred substrate for mtFabD. In order to provide further insight into the structure-function relationship of mtFabD, different mutant proteins were generated. All mutations (Q9A, R116A, H194A, Q243A, S91T, and S91A) completely abrogated MCAT activity in vitro, thus underlining the importance of these residues in transacylation. The generation and characterization of the AcpM forms and mtFabD opens the way for further studies relating to fatty acid and mycolic acid biosynthesis to be explored in M. tuberculosis. Since a specific type of FabD is found in mycobacterial species, it represents an attractive new drug target waiting to be exploited.

Urease of Corynebacterium glutamicum: organization of corresponding genes and investigation of activity.

The Corynebacterium glutamicum genes encoding urease were isolated and sequenced. While ureA, ureB and ureC are encoding structural subunits of urease, ureE, ureF, ureG and ureD are encoding accessory proteins. As deduced from DNA sequence analyses, the ure genes are transcriptionally coupled, this was proven by RT-PCR at least for ureABC. Gene disruption experiments revealed that both structural (UreC) and accessory proteins (UreD) are indispensable for urease activity and growth on urea. Urease activity was determined in different Corynebacterium species after growth in various media. While the regulation patterns observed revealed species-specific differences, in general urease activity is induced upon nitrogen starvation. As in mycobacteria, in corynebacteria urease activity was highest in a pathogenic species and might also play a role in host-pathogen interaction.