Nutritional Requirements and Nitrogen-Dependent Regulation of Proteinase Activity of Lactobacillus helveticus CRL 1062

The nutritional requirements of Lactobacillus helveticus CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of L. helveticus CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM. The growth rate and the synthesis of their cell membrane-bound serine proteinases, but not of their intracellular leucyl-aminopeptidases, were influenced by the peptide content of the medium. The highest proteinase levels were found during cell growth in basal SCDM, while the synthesis of this enzyme was inhibited in SCDM supplemented with Casitone, Casamino Acids, or β-casein. Low-molecular-mass peptides (<3,000 Da), extracted from Casitone, and the dipeptide leucylproline (final concentration, 5 mM) play important roles in the medium-dependent regulation of proteinase activity. The addition of the dipeptide leucylproline (5 mM) to SCDM reduced proteinase activity by 25%.     

Characterization of a Cell Membrane-Associated Proteinase from Lactobacillus helveticus CRL 581

The production of a proteinase from Lactobacillus helveticus CRL 581 was studied. The highest specific activity was found at the early exponential growth phase of cells cultured in milk. The lowest levels of proteinase were detected in MRS broth, while in the casein–yeast extract–glucose broth enzyme production increased gradually during the fermentation and reached maximal values at the stationary phase. The proteinase, found to be associated with the cell membrane fraction, hydrolyzed β-casein more rapidly than α-casein. The enzyme was not released from washed cells in the presence or absence of calcium, which suggests that the enzyme did not undergo self-digestion. Received: 28 January 1997 / Accepted: 8 March 1997     

Characterisation of a cell-envelope proteinase from Lactobacillus helveticus

The cell-envelope proteinase from Lactobacillus helveticus CRL 1062 was detected in the cell membrane fraction. The enzyme remained associated with the cells even after treatment with lysozyme and was not released from washed cells in absence of calcium. The proteinase was maximally active at pH 6.5–7.0 and 42°C and hydrolysed - and -caseins at different rates. Activity was inhibited (98%) by 1 mM PMSF, suggesting it was a serine-type protease.     

Purification and characterization of the mature,membrane-associated cell-envelope proteinase of Lactobacillus helveticus L89

Lactobacillus helveticus L89 possesses a cell-envelope proteinase (Lb-CEP) that is biochemically and genetically related to that of the lactococci (Lc-CEP). The in-situ proteinase is resistant to autoproteolysis and remains associated with the membrane during lysozyme treatment of cells and subsequent mechanical disruption of the treated cells. The proteinase was purified from isolated membranes by a procedure that preserves the complete in-situ proteinase (mature proteinase) assumed to be the N-terminally processed translation product including the membrane anchor: its monomer molecular mass is approximately 180 kDa. The purified enzyme appeared to be more stable towards heat than hitherto known related, but C-terminally truncated cell-envelope proteinases of lactobacilli and lactococci, which were released from the cells by autoproteolysis. On the basis of its specificity towards caseins, towards the _sl-casein-(1-23)-fragment and towards two differently charged chromophoric peptides, the proteinase was recognized as an (Lb-)CEP_I/III mixed-type variant different from those identified so far among the lactococcal proteinases.Correspondence to: F. A. Exterkate     

Molecular cloning and sequence analysis of a gene encoding an extracellular proteinase from Lactobacillus helveticus CP790

A 2.6-kilobase HaeIII DNA fragment corresponding to an extracellular proteinase gene (prtY) was cloned from chromosomal DNA of Lactobacillus helveticus CP790 in Escherichia coli using a pKK223-3 vector. The transformant expressed a 48-kDa protein that reacts with monoclonal antibodies specific to the proteinase and seemed to be a pre-proproteinase, but had no proteolytic activity. About 1.6 kilobases of the 2.6-kilobase DNA fragment, which contained the complete gene for the proteinase was sequenced. Sequence analysis found an open reading frame with a capacity to encode a protein of 449 amino acids. The coding region contained a Gram-positive-type signal peptide of 30 amino acids. The N-terminal sequences of the proproteinase and the mature proteinase have been observed in the polypeptide at position + 31 and + 38. The putative amino acid sequence showed a significant similarity to a surface layer protein of L. helveticus and Lactobacillus acidophilus in the amino terminal signal sequence and carboxyl terminus.     

Fermentation of quinoa and wheat slurries by Lactobacillus plantarum CRL 778: proteolytic activity

Quinoa fermentation by lactic acid bacteria (LAB) is an interesting alternative to produce new bakery products with high nutritional value; furthermore, they are suitable for celiac patients because this pseudo-cereal contains no gluten. Growth and lactic acid production during slurry fermentations by Lactobacillus plantarum CRL 778 were greater in quinoa (9.8 log?cfu/mL, 23.1 g/L) than in wheat (8.9 log?cfu/mL, 13.9 g/L). Lactic fermentation indirectly stimulated flour protein hydrolysis by endogenous proteases of both slurries. However, quinoa protein hydrolysis was faster, reaching 40–100 % at 8 h of incubation, while wheat protein hydrolysis was only 0–20 %. In addition, higher amounts of peptides (24) and free amino acids (5 g/L) were determined in quinoa compared to wheat. Consequently, greater concentrations (approx. 2.6-fold) of the antifungal compounds (phenyllactic and hydroxyphenyllactic acids) were synthesized from Phe and Tyr in quinoa by L. plantarum CRL 778, an antifungal strain. These promising results suggest that this LAB strain could be used in the formulation of quinoa sourdough to obtain baked goods with improved nutritional quality and shelf life, suitable for celiac patients.     

Zymogram and Preliminary Characterization of Lactobacillus helveticus Autolysins

The autolysins of Lactobacillus helveticus ISLC5 were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymogram). By using lyophilized Micrococcus luteus cells or heated whole cells of L. helveticus ISLC5 (0.2% [wt/vol]) as a substrate, several lytic activities were detected in the whole-cell SDS extract of strain ISLC5 (i) one activity at 42.4 kDa, which was named autolysin A, and (ii) six other activities having very similar molecular weights (29.1, 29.6, 30, 30.8, 31.7, and 32.8 kDa), which were named autolysins B (B1 through B6, respectively). As regards the temporal distribution of the enzymes, autolysins A and B were detected in the cells harvested from the beginning of the exponential growth phase. Autolysin A appeared to be associated only with viable cells, whereas the autolysins B remained associated with the cell envelope several days after the complete loss of culture viability. When SDS-treated walls of L. helveticus ISLC5 were used as a substrate, a supplementary lytic activity appeared at 37.5 kDa; it was considered a peptidoglycan hydrolase, since it was not able to induce lysis of whole-cell substrate. The autolysins of 30 other strains of L. helveticus from various geographical origins were also analyzed by zymogram; all the activity profiles obtained were similar to that of strain ISLC5 in terms of the number of lytic bands and their apparent molecular weights. Only the relative intensities of the lytic bands corresponding to autolysins A and B were variable depending on the strains. This observation suggested that autolysins are highly conserved enzymes. A concentrated crude lysate of the virulent bacteriophage 832-B1 infecting L. helveticus was also analyzed by zymogram; one lytic activity with an apparent molecular weight of 31.7 kDa, very close to the weights of the autolysins B, was observed. Finally, the autolysins of L. helveticus ISLC5 were successfully extracted from whole cells by using a 1 M lithium chloride solution; they were partially purified by precipitation, selective resolubilization, and gel filtration chromatography, which led to a 20-fold increase in specific activity.     

Cholesterol assimilation and biotransformation by Lactobacillus helveticus

Lactobacillus helveticus, grown at 37°C in MRS medium supplemented with 3 mM cholesterol, assimilated all the cholesterol in 42 h having 68 U mg⁻¹ of intracellular cholesterol oxidase activity. The strain transformed 1 g cholesterol to 0.05 g of androsta-1, 4-diene-3, 17-dione and 0.04 g of androst-4-ene-3, 17 dione within 48 h at 37°C with extracellular cholesterol oxidase activity at 12 U mg⁻¹ and intracellular oxidase at 0.5 U mg⁻¹.     

Administration of Lactobacillus fermentum CRL1446 increases intestinal feruloyl esterase activity in mice

Aims: To evaluate the effect of oral administration of Lactobacillus fermentum CRL1446 on the intestinal feruloyl esterase (FE) activity and oxidative status of mice. Methods and Results: Adult Swiss albino mice received Lact. fermentum CRL1446 at the doses 10⁷ and 10⁹ cells per day per mouse for 2, 5, 7 and 10 days. Intestinal FE activity, intestinal microbiota counts, plasmatic thiobarbituric acid‐reactive substances (TBARS) percentage and glutathione reductase (GR) activity were determined. Mice that received Lact. fermentum CRL1446 at the dose 10⁷ cells per day for 7 days showed a twofold increase in total intestinal FE activity, compared to the nontreated group. In large intestine content, FE activity increased up to 6·4 times. No major quantitative changes in colonic microbiota were observed in treated animals. Administration of this strain produced an approx. 30–40% decrease in the basal levels of plasmatic TBARS and an approx. twofold increase in GR activity from day 5 of feeding with both doses. Conclusions: Oral administration of Lact. fermentum CRL1446 to mice increases total intestinal FE activity, decreases the basal percentage of plasmatic lipoperoxides and increases GR activity. Significance and Impact of the Study: Lactobacillus fermentum CRL1446 could be orally administered as a dietary supplement or functional food for increasing the intestinal FE activity to enhance the bioavailability of ferulic acid, thus improving oxidative status.     

Nutritional regulation of proteinase production in the fungus, Tritirachium album

The fungus, Tritirachium album, produces a number of proteinases under proper conditions. We have studied the nutritional regulation mechanisms for proteinase production in the mold, i.e. the effects of carbon and nitrogen sources, and the influence of starvation. Proteinase production was induced when the nitrogen source was an exogenous protein or peptide, such as peptones or yeast extract. The production rate was affected by the amount of available inducing substrate. Inorganic nitrogen compounds, i.e., ammonium or nitrate salts, had a repressing effect on the production. Production was not induced if a detectable concentration of glucose or sucrose was present in the medium. Starvation did not trigger proteinase production. Journal of Industrial Microbiology & Biotechnology (2000) 24, 369–373. Received 20 January 1999/ Accepted in revised form 06 March 2000     

Purification and Characterization of Invertase from Lactobacillus reuteri CRL 1100

The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa. The enzyme was stable below 45°C over a wide pH range (4.5–7.0) with maximum activity at pH 6.0 and 37°C. The invertase activity was significantly inhibited by bivalent metal ions (Ca⁺⁺, Cu⁺⁺, Cd⁺⁺, and Hg⁺⁺), β-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid. The enzyme was purified 32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17%. The K _m and V_max values for sucrose were 6.66 mM and 0.028 μmol/min, respectively. An invertase is purified and characterized for the first time in Lactobacillus, and it proved to be a β-fructofuranosidase. Received: 13 August 1999 / Accepted: 15 September 1999     

Complete genome sequence of Lactobacillus helveticus H10

Lactobacillus helveticus strain H10 was isolated from traditional fermented milk in Tibet, China. We sequenced the whole genome of strain H10 and compared it to the published genome sequence of Lactobacillus helveticus DPC4571.     

Catalytic components of proteasomes and the regulation of proteinase activity

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the and subunits of the simpler proteasome isolated fromThermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that -type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Prel, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these -type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulatedin vivo. Our recent studies have shown that in animal cells at least two proteasome subunits can undergo phosphorylation, the level of which is likely to be important for determining proteasome localization, activity or ability to form larger complexes. In addition, we have isolated two isoforms of the 26S proteinase.     

Fast and slow milk-coagulating variants of Lactobacillus helveticus HLM 1

Slow milk-coagulating variants were isolated from Lactobacillus helveticus HLM 1, a fast strain which coagulates milk in 16 h at 42 degrees C. Variants were isolated after subculturing in reconstituted skim milk or complex broth media. Analysis of plasmid content revealed that in slow variants a 3.5-megadalton plasmid was missing.     

Cryptic plasmids from Lactobacillus helveticus and their evolutionary relationship

Abstract Three different cryptic plasmids from Lactobacillus helveticus have been identified and their DNA sequences determined. Analyses and comparisons of their primary structures revealed stretches of DNA with considerable homology. Thus, large portions of the plasmid non-coding sequences were conserved at 80–90% identity between the different plasmids identified so far in L. helveticus . Nevertheless, different plasmids found in a same host strain utilise different genes of replication, probably acquired during evolution from different replicons from Gram-positive bacterial origins. A remnant structure of such a possible genetic integration of a foreign replication gene into one of the plasmids of L. helveticus was identified.     

Heterogeneity of Putative Surface Layer Proteins in Lactobacillus helveticus

The S-layer-encoding genes of 21 Lactobacillus helveticus strains were characterized. Phylogenetic analysis based on the identified S-layer genes revealed two main clusters, one which includes a sequence similar to that of the slpH1 gene of L. helveticus CNRZ 892 and a second cluster which includes genes similar to that of prtY. These results were further confirmed by Southern blot hybridization. This study demonstrates S-layer gene variability in the species L. helveticus.     

Heterogeneity of putative surface layer proteins in Lactobacillus helveticus

The S-layer-encoding genes of 21 Lactobacillus helveticus strains were characterized. Phylogenetic analysis based on the identified S-layer genes revealed two main clusters, one which includes a sequence similar to that of the slpH1 gene of L. helveticus CNRZ 892 and a second cluster which includes genes similar to that of prtY. These results were further confirmed by Southern blot hybridization. This study demonstrates S-layer gene variability in the species L. helveticus.