Properties of Alanine Dehydrogenase and Aspartase from Propionibacterium freudenreichii subsp. shermanii

During lactate fermentation by Propionibacterium freudenreichii subsp. shermanii ATCC 9614, the only amino acid metabolized was aspartate. After lactate exhaustion, alanine was one of the two amino acids to be metabolized. For every 3 mol of alanine metabolized, 2 mol of propionate, 1 mol each of acetate and CO₂, and 3 mol of ammonia were formed. The specific activity of alanine dehydrogenase was 0.08 U/mg of protein during lactate fermentation, and it increased to 0.9 U/mg of protein after lactate exhaustion. Alanine dehydrogenase and aspartase, key enzymes in the metabolism of alanine and aspartate, respectively, were partially purified, and some of their properties were studied. Alanine dehydrogenase had a pH optimum of 9.2 to 9.6 and high K_m values for both NAD⁺ (1 to 4 mM) and alanine (7 to 20 mM). Activity was inhibited by low concentrations of pyruvate and NADH. The pH optimum of aspartase decreased from ~7.5 to ~6.4 when the MgCl₂ and aspartate concentrations were decreased. Plots of aspartate concentration versus activity showed either hyperbolic or sigmoidal kinetics (interaction coefficient, up to a value of 3.1), depending on pH and MgCl₂ concentration. MgCl₂ was either an activator or an inhibitor, depending on pH and its concentration. Aspartase activity was inhibited by low concentrations of fumarate. The properties of alanine dehydrogenase and aspartase are consistent with the finding that aspartate is metabolized during lactate fermentation, while alanine is only fermented after lactate exhaustion and then at a slow rate.     

First evidence of lysogeny in Propionibacterium freudenreichii subsp. shermanii

Dairy propionic acid bacteria, particularly the species Propionibacterium freudenreichii, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of P. freudenreichii was readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in P. freudenreichii.     

First Evidence of Lysogeny in Propionibacterium freudenreichii subsp. shermanii

Dairy propionic acid bacteria, particularly the species Propionibacterium freudenreichii, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of P. freudenreichii was readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in P. freudenreichii.     

13C-NMR study of lactate metabolism in Propionibacterium freudenreichii subsp. shermanii

The adaptation to osmotic stress in Propionibacterium freudenreichii subsp. shermanii was investigated by using natural-abundance ¹³C nuclear magnetic resonance spectroscopy. Cells incubated either in a standard laboratory medium or in a medium designed to simulate the physicochemical conditions of Swiss-type cheese were found to accumulate different levels of osmotic-stress-protectant molecules. Proline, betaine, trehalose and glutamate were found simultaneously. Moreover, two types of polysaccharides were in evidence in this strain. Lactate catabolism was not mainly directed towards cell growth requirements and organic acid production but also towards biosynthesis of osmolytes requested for adaptation in a cheese environment. The possible involvement of such type of metabolite accumulation in the main cheese-ripening bacteria in Swiss-type cheeses is discussed.     

Metabolism of Aspartate by Propionibacterium freudenreichii subsp. shermanii: Effect on Lactate Fermentation

More than 90% of the aspartate in a defined medium was metabolized after lactate exhaustion such that 3 mol of aspartate and 1 mol of propionate were converted to 3 mol of succinate, 3 mol of ammonia, 1 mol of acetate, and 1 mol of CO₂. This pathway was also evident when propionate and aspartate were the substrates in complex medium in the absence of lactate. In complex medium with lactate present, about 70% of the aspartate was metabolized to succinate and ammonia during lactate fermentation, and as a consequence of aspartate metabolism, more lactate was fermented to acetate and CO₂ than was fermented to propionate. The conversion of aspartate to fumarate and ammonia by the enzyme aspartase and subsequent reduction of fumarate to succinate occurred in the five strains of Propionibacterium freudenreichii subsp. shermanii studied. The ability to metabolize aspartate in the presence of lactate appeared to be related to aspartase activity. The specific activity of aspartase increased during and after lactate utilization, and the levels of this enzyme were lower in cells grown in defined medium than levels in those cells grown in complex medium. Under the conditions used, no other amino acids were readily metabolized in the presence of lactate. The possibility that aspartate metabolism by propionibacteria in Swiss cheese has an influence on CO₂ production is discussed.     

Citrate Cycle Intermediates in the Metabolism of Aspartate and Lactate by Propionibacterium freudenreichii subsp. shermanii

Propionibacterium freudenreichii subsp. shermanii metabolized 7 mol of aspartate to 6 mol of succinate, 4 mol of CO₂, and 7 mol of ammonia. When lactate, sparged with 100% CO₂, was fermented at pH 5.5, unexpectedly high ratios of propionate to acetate were obtained (i.e., 3.2 to 3.8:1). Citrate cycle intermediates may be involved in these fermentations.     

Effect of cobalamin derivatives on DNA synthesis in cells of Propionibacterium freudenreichii subsp. shermanii

The increase of DNA-synthesis rate (according incorporation [8-14C]adenine) in B12-deficient cells Propionibacterium shermanii as a result of different cobalamines adding into the cell suspension including metoxyethyladenile analog of adenozilcobalamin and some components of vitamin B12 molecule has been found. The DNA-synthesis rate in B12-deficient cells is nearly twice lower as compared with one in B12-normal cells. Considerable stimulative effect (80-100%) was provided with coenzyme forms of cobalamin. The data confirm the participation of vitamin B12 in DNA-synthesis in Propionibacterium cells.     

Lipase and Esterase Activities of Propionibacterium freudenreichii subsp. freudenreichii

The lipase and esterase activities of eight strains of dairy Propionibacterium freudenreichii subsp. freudenreichii were studied. A lipase activity was detected on whole cells and in the culture supernatant. The highest activity was expressed at 45°C and pH 6.8. An esterase activity was also detected in the culture medium. The electrophoresis of the intracellular fractions of the cells revealed from three to six different esterase activities. Two esterases were common to all the strains. The substrate specificity was dependent on each esterase, but no activity was revealed, in our experimental conditions, on ester substrates with a chain length longer than that of butyrate.     

Inhibition of Induced Mutagenesis in Salmonella typhimurium by the Protein of Propionibacterium freudenreichii subsp. shermanii

Culture liquid and cells of Propionibacterium freudenreichii subsp. shermanii VKM-103 exerted a strong antimutagenic effect on mutations induced by 4-nitroquinoline-1-oxide, N-methyl-N′-nitro-N′-nitrosoguanidine, sodium azide (base pair substitutions) and 9-aminoacridine (frameshift mutations). No inhibitory effect was observed against mutagenesis induced by 2-nitrofluorene (frameshift mutations). The highest antimutagenic activity was found in the culture liquid of cells grown for 24 h. Acetic and propionic acids of the culture liquid produced by propionibacteria made no observable contribution to the antimutagenicity. Antimutagenic activity of the culture liquid was considerably reduced by protease treatment and by heating at 92°C for 10 min. Upon dialysis, the culture liquid lost almost all of its inhibitory activity. Cell wash solution also contained high antimutagenic activity which was lost upon protease treatment and dialysis. According to the exclusion limit of the dialysis bag, the molecular weight of the antimutagenic factor, presumably a protein, is less than 1.5 kDa. In addition, the cells of P. shermanii were capable of binding or modifying the mutagens, thereby decreasing their mutagenicity.     

Utilization of Lactate Isomers by Propionibacterium freudenreichii subsp. shermanii: Regulatory Role for Intracellular Pyruvate

Five strains of Propionibacterium freudenreichii subsp. shermanii utilized the l-(+) isomer of lactate at a faster rate than they did the d-(-) isomer when grown with a mixture of lactate isomers under a variety of conditions. ATCC 9614, grown anaerobically in defined medium containing 160 mM dl-lactate, utilized only 4 and 15% of the d-(-)-lactate by the time 50 and 90%, respectively, of the l-(+)-lactate was used. The intracellular pyruvate concentration was high (>100 mM) in the initial stages of lactate utilization, when either dl-lactate or the l-(+) isomer was the starting substrate. The concentration of this intermediate dropped during dl-lactate fermentation such that when only d-(-)-lactate remained, the concentration was <20 mM. When only the d-(-) isomer was initially present, a similar relatively low concentration of intracellular pyruvate was present, even at the start of lactate utilization. The NAD-independent lactate dehydrogenase activities in extracts showed different kinetic properties with regard to pyruvate inhibition, depending upon the lactate isomer present. Pyruvate gave a competitive inhibitor pattern with l-(+)-lactate and a mixed-type inhibitor pattern with d-(-)-lactate. It is suggested that these properties of the lactate dehydrogenases and the intracellular pyruvate concentrations explain the preferential use of the l-(+) isomer.     

Propionic acid fermentation of glycerol and glucose by Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp.shermanii

A comparative study was carried out in anaerobic batch cultures on 20 g/l of either glycerol or glucose using two propionibacteria strains, Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp. shermanii. In all cases, fermentation end-products were the same and consisted of propionic acid as the major product, acetic acid as the main by-product and two minor metabolites, n-propanol and succinic acid. Evidence was provided that greater production of propionic acid by propionibacteria was obtained with glycerol as carbon and energy sources. P. acidipropionici showed higher efficiency in glycerol conversion to propionic acid with a faster substrate consumption (0.64 g l⁻¹ h⁻¹) and a higher propionic acid production (0.42 g l⁻¹ h⁻¹ and 0.79 mol/mol). The almost exclusive production of propionic acid from glycerol by this bacterium suggested an homopropionic tendency of this fermentation. Acetic acid final concentration was two times lower on glycerol (2 g/l) than on glucose (4 g/l) for both micro-organisms. P. freudenreichii ssp. shermanii exhibited a glycerol fermentation pattern typical of non-associated glycerol-consumption-product formation. This could indicate a particular metabolism for P. freudenreichii ssp. shermanii oriented towards the production of other specific components. These results tend to show that glycerol could be an excellent alternative to conventional carbon sources such as carbohydrates for propionic acid production. Received: 21 May 1999 / Accepted: 1 November 1999     

Biosynthesis of vitamin B12 by Propionibacterium freudenreichii subsp. shermanii ATCC 13673 using liquid acid protein residue of soybean as culture medium

Vitamin B12 deficiency still persists, mainly caused by low intake of animal food products affecting vegetarians, vegans, and populations of underdeveloped countries. In this study, we investigate the biosynthesis of vitamin B12 by potential probiotic bacterium using an agroindustry residue, the liquid acid protein residue of soybean (LAPRS), as a low-cost, animal derivate-free alternative culture medium. Cultures of Propionibacterium freudenreichii subsp. shermanii ATCC 13673 growing in LAPRS for vitamin B12 biosynthesis were studied using the Plackett–Burman experimental approach, followed by a central composite design 2² to optimize the concentration of significant variables. We also performed a proteolytic treatment of LAPRS and evaluated the optimized–hydrolyzed medium influence on the microbial growth and metabolism in shaker flask and bioreactor experiments. In this all-plant source medium, P. freudenreichii subsp. shermanii produced high concentrations of cells and high amounts of vitamin B12 (0.6 mg/g cells) after process optimization. These results suggest the possibility of producing vitamin B12 by a potential probiotic bacterium in a very cheap, animal derivate-free medium to address the needs of specific population groups, at the same time reducing the production costs of this essential vitamin.     

Reduction of linoleic acid inhibition in production of conjugated linoleic acid by Propionibacterium freudenreichii ssp. shermanii

A method for the production of conjugated linoleic acid (CLA) from linoleic acid (LA) using growing cultures of Propionibacterium freudenreichii ssp. shermanii JS was developed. The growth inhibitory effect of LA was eliminated by dispersing it in a sufficient concentration of polyoxyethylene sorbitan monooleate detergent. For the whey permeate medium used, the optimum LA:detergent ratio was 1:15 (w/w). As a result, the cultures tolerated at least 1000 microg x mL(-1) LA, which was converted to CLA with 57%-87% efficiency. The cis-9, trans-11 and trans-9, cis-11 isomers constituted 85%-90% of the CLA produced. The feasibility of the method was demonstrated also in de Man Rogosa-Sharpe (MRS) broth.     

Interactions between Pyruvate and Lactate Metabolism in Propionibacterium freudenreichii subsp. shermanii: In Vivo 13C Nuclear Magnetic Resonance Studies

In vivo ¹³C nuclear magnetic resonance spectroscopy was used to elucidate the pathways and the regulation of pyruvate metabolism and pyruvate-lactate cometabolism noninvasively in living-cell suspensions of Propionibacterium freudenreichii subsp. shermanii. The most important result of this work concerns the modification of fluxes of pyruvate metabolism induced by the presence of lactate. Pyruvate was temporarily converted to lactate and alanine; the flux to acetate synthesis was maintained, but the flux to propionate synthesis was increased; and the reverse flux of the first part of the Wood-Werkman cycle, up to acetate synthesis, was decreased. Pyruvate was consumed at apparent initial rates of 148 and 90 μmol · min⁻¹ · g⁻¹ (cell dry weight) when it was the sole substrate or cometabolized with lactate, respectively. Lactate was consumed at an apparent initial rate of 157 μmol · min⁻¹ · g⁻¹ when it was cometabolized with pyruvate. P. shermanii used several pathways, namely, the Wood-Werkman cycle, synthesis of acetate and CO₂, succinate synthesis, gluconeogenesis, the tricarboxylic acid cycle, and alanine synthesis, to manage its pyruvate pool sharply. In both types of experiments, acetate synthesis and the Wood-Werkman cycle were the metabolic pathways used most.     

Biosynthesis of vitamin B12. Some properties of the 5,6-dimethylbenzimidazole-forming system of Propionibacterium freudenreichii and Propionibacterium shermanii

1. Homogenates of Propionibacterium freudenreichii transform riboflavin into 5,6-dimethylbenzimidazole. This process is stimulated by nicotinamide. Homogenates of Propionibacterium shermanii form only small amounts of 5,6-dimethylbenzimidazole from riboflavin in the absence of nicotinamide, but also form appreciable amounts in the presence of nicotinamide. 2. The stimulation of the 5,6-dimethylbenzimidazole-forming system by nicotinamide shows a lag phase which is abolished by preincubation of the homogenate with nicotinamide. Since no lag phase is observed when nicotinamide is replaced by nicotinate, nicotinate seems to be the true stimulating agent. These observations are in agreement with the fact that nicotinamide is rapidly split to nicotinate in homogenates of P. freudenreichii. 3. The 5,6-dimethylbenzimidazole-forming homogenate system is only active at a high buffer concentration (0.3--0.5 M) and in the presence of oxygen. The system has a pronounced oxygen optimum. 4. Flavin mononucleotide and flavin-adenine dinucleotide are better substrates for the 5,6-dimethylbenzimidazole-forming homogenate system than riboflavin. But with [1'-14C]riboflavin as substrate the specific radioactivity of 5,6-dimethylbenzimidazole is higher than the specific radioactivity of flavin--adenine dinucleotide and lower than the specific radioactivie substrate for the formation of 5,6-dimethylbenzimidazole. 5. A tentative reaction sequence for the transformation of flavin mononucleotide into 5,6-dimethylbenzimidazole is discussed.     

In vivo 13C NMR study of the bidirectional reactions of the Wood-Werkman cycle and around the pyruvate node in Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici

This study used in vitro 13C NMR spectroscopy to directly examine bidirectional reactions of the Wood-Werkman cycle involved in central carbon metabolic pathways of dairy propionibacteria during pyruvate catabolism. The flow of [2-13C]pyruvate label was monitored on living cell suspensions of Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici under acidic conditions. P. shermanii and P. acidipropionici cells consumed pyruvate at apparent initial rates of 161 and 39 micromol min(-1) g(-1) (cell dry weight), respectively. The bidirectionality of reactions in the first part of the Wood-Werkman cycle was evident from the formation of intermediates such as [3-13C]pyruvate and [3-13C]malate and of products like [2-13C]acetate from [2-13C]pyruvate. For the first time alanine labeled on C2 and C3 and aspartate labeled on C2 and C3 were observed during [2-13C]pyruvate metabolism by propionibacteria. The kinetics of aspartate isotopic enrichment was evidence for its production from oxaloacetate via aspartate aminotransferase. Activities of a partial tricarboxylic acid pathway, acetate synthesis, succinate synthesis, gluconeogenesis, aspartate synthesis, and alanine synthesis pathways were evident from the experimental results.     

Interactions between pyruvate and lactate metabolism in Propionibacterium freudenreichii subsp. shermanii: in vivo (13)C nuclear magnetic resonance studies

In vivo (13)C nuclear magnetic resonance spectroscopy was used to elucidate the pathways and the regulation of pyruvate metabolism and pyruvate-lactate cometabolism noninvasively in living-cell suspensions of Propionibacterium freudenreichii subsp. shermanii. The most important result of this work concerns the modification of fluxes of pyruvate metabolism induced by the presence of lactate. Pyruvate was temporarily converted to lactate and alanine; the flux to acetate synthesis was maintained, but the flux to propionate synthesis was increased; and the reverse flux of the first part of the Wood-Werkman cycle, up to acetate synthesis, was decreased. Pyruvate was consumed at apparent initial rates of 148 and 90 micromol. min(-1). g(-1) (cell dry weight) when it was the sole substrate or cometabolized with lactate, respectively. Lactate was consumed at an apparent initial rate of 157 micromol. min(-1). g(-1) when it was cometabolized with pyruvate. P. shermanii used several pathways, namely, the Wood-Werkman cycle, synthesis of acetate and CO(2), succinate synthesis, gluconeogenesis, the tricarboxylic acid cycle, and alanine synthesis, to manage its pyruvate pool sharply. In both types of experiments, acetate synthesis and the Wood-Werkman cycle were the metabolic pathways used most.     

Purification of methylmalonyl-CoA mutase from Propionibacterium shermanii using affinity chromatography.

A novel procedure for the purification of methylmalonyl-CA mutase from Propionibacterium shermanii has been described which employs affinity chromatography on a column of immobilized vitamin B-12 linked covalently to Sepharose. The method has the advantage of being simple and rapid, thus enabling the purification of the enzyme to near homogeneity with good yields.     

Mechanism of the ring contraction process in vitamin B12 biosynthesis by the anaerobe Propionibacterium shermanii under aerobic conditions

The mechanism of the ring contraction process during vitamin B(12) biosynthesis by the anaerobe Propionibacterium shermanii was investigated under both aerobic and anaerobic conditions by means of feeding experiments with delta-amino[1-(13)C]levulinic acid (a biosynthetic intermediate of tetrapyrrole) and delta-amino[1-(13)C,1,1,4-(18)O(3)]levulinic acid in combination with (13)C-NMR spectroscopy. We showed that the characteristic mechanism of the ring contraction process (the generation of precorrin-3x from formation of the gamma-lactone from the ring A acetate group at C1 and hydroxylation at C20 by molecular oxygen catalyzed by CobG, and the migration of ring D by cleavage of the carbon-oxygen bond at C1 of precorrin-3x) in the aerobe Pseudomonas denitrificans was not seen in P. shermanii under aerobic conditions, and the mechanism of the ring contraction process in P. shermanii was the same irrespective of the presence or absence of oxygen.