Production of Cell-Bound Proteinase by Lactobacillus bulgaricus and its Location in the Bacterial Cell

Lactobacillus bulgaricus NCDO 1489 produced a single, cell-bound proteinase during growth on nutrient medium at 45°C. Proteinase activity was optimal at 45–50°C and pH 5.2–5.8. and was inhibited by chelating agents. The enzyme was mainly associated with the cell envelope but could be liberated from cells under conditions favouring autolysis or by treatment of the cells with lysozyme. Its relation to the growth of the organism in milk and possible role in formation of fermented milk products are discussed.     

Effect of environmental pH on fermentation balance of Lactobacillus bulgaricus.

When Lactobacillus bulgaricus NLS-4 was grown anaerobically in continuous culture with limiting glucose, a shift in the pH of the medium from the acidic to the alkaline range caused this normally homofermentative bacterium to catabolize glucose in a heterofermentative fashion. The change in the nature of the fermentation was accompanied by a decrease in lactate dehydrogenase biosynthesis in alkaline conditions. The lactate dehydrogenase from this organism did not require fructose 1,6-diphosphate or manganese ions (Mn2+) for catalytic activity. Involvement of the phosphoroclastic split in the pyruvate conversion in an alkaline environment was also confirmed. The high lactate dehydrogenase synthesis in acidic medium together with the participation of the phosphoroclastic split under alkaline conditions may explain the shift from homolactic to heterolactic fermentation of L. bulgaricus NLS-4 with the change of environmental pH.     

Selective enumeration of Lactobacillus casei from yogurts and fermented milk drinks

A selective medium (LC agar) was developed for enumeration of Lactobacillus casei populations from commercial yogurts and fermented milk drinks that may contain strains of yogurt bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), probiotic bacteria (Lactobacillus acidophilus and bifidobacteria) and L. casei. Appropriate dilutions were pour-plated in specially formulated LC agar acidified to pH 5.1 and the plates incubated at 27°C for 72 to 96 h under anaerobic conditions. Growth of S. thermophilus was prevented by adjusting pH to 5.1. L. delbrueckii ssp. bulgaricus did not ferment ribose as the carbon source, as a result the organisms did not form colonies. L. acidophilus formed colonies on MRS-ribose agar; however, this organism did not grow in the specially formulated LC agar containing ribose. Similarly, Bifidobacterium spp. did not form colonies in LC agar. L. casei formed colonies on LC agar. © Rapid Science Ltd. 1998     

Effects of various sugars added to growth and drying media upon thermotolerance and survival throughout storage of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus

The aim of this research effort was to investigate the role of various sugar substrates in the growth medium upon thermotolerance and upon survival during storage after freeze-drying of Lactobacillus bulgaricus. Addition of the sugars tested to the growth medium, and of these and sorbitol to the drying medium (skim milk) was investigated so as to determine whether a relationship exists between growth and drying media, in terms of protection of freeze-dried cells throughout storage. The lowest decrease in viability of L. bulgaricus cells after freeze-drying was obtained when that organism was grown in the presence of mannose. However, L. bulgaricus clearly survived better during storage when cells had been grown in the presence of fructose, lactose or mannose rather than glucose (the standard sugar in the growth medium). A similar effect could not be observed in terms of thermotolerance; in this case, the growth medium supplemented with lactose was found to yield cells bearing the highest heat resistance. Supplementation of the drying medium with glucose, fructose, lactose, mannose or sorbitol led in most cases to enhancement of protection during storage, to a degree that was growth medium-dependent.     

Formation and properties of L-glutaminase and L-asparaginase activities in Pichia polymorpha

An ascogenous yeast with high potentialities for L-glutaminase and L-asparaginase formation was isolated from Egyptian soils by the application of the culture enrichment method. The organism, identified as Pichia polymorpha, was obtained through the enrichment of soil samples with a simple medium containing 0.5% L-glutaminase as a major carbon and nitrogen source at low pH values. The amidase activities were produced constitutively on a variety of media irrespective of the presence of their substrates in the growth medium. Assays of enzyme activity have revealed that optimum pH values for L-glutamine and L-asparagine hydrolysis are 6.0 and 6.7, respectively. The L-asparaginase activity of the cells was heat-stable for at least 10 minutes at 60 degrees C. The enzyme exhibited apparent Km of 1.37 x 10(-2) M and 1.95 x 10(-2) M for L-asparagine and L-glutamine, respectively. No metal requirement were detected for the amidase activities of the organism under study.     

Factors affecting production and activity of phospholipase C by Yersinia enterocolitica

The production of phospholipase C by Yersinia enterocolitica strain SG was optimum at 37 degrees C at pH 6.5. No enzyme activity could be detected when the organism was grown at extreme pH values (pH greater than 8.5 or less than 5.0). The enzyme production was maximum when the organism was grown under static conditions in TSB medium. All solvents and salts inhibited the enzyme activity, whereas loss of activity was 95% in presence of methanol (20%) and 99% in presence of sodium azide (0.2 mol/l). The enzyme activity was increased twofold in the presence of cysteine and decreased by 98% in the presence of sodium perchlorate (0.2 mol/l).     

Effect of the pH of growth on the survival of Lactobacillus delbrueckii subsp. bulgaricus to stress conditions during spray-drying

AIMS: The aim of this study was to optimize survival of Lactobacillus delbrueckii subsp. bulgaricus during spray-drying and subsequent storage through optimizing the pH of growth conditions. METHODS AND RESULTS: Cell concentrates previously grown without or with pH controlled were spray-dried and stored at 20 degrees C and heat treated at 57 degrees C. Cells grown under noncontrolled pH were more resistant to both drying and heating than cells grown under controlled pH but no significant differences were observed during storage. The intracellular proteins profile of cells grown under both conditions was studied by two-dimensional SDS-polyacrylamide gel electrophoresis. Eight proteins were identified using automated mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data acquisition. Of the identified proteins, only cochaperonin GroES corresponded to a known heat shock protein (HSP). The other proteins identified are proteins involved in glycolysis. For cells grown under noncontrolled pH the expression of the Hsp70, GroES and GroEL, measured by Western blotting, was enhanced. CONCLUSIONS: The higher resistance of cells grown under noncontrolled pH correlates with the enhanced production of heat shock proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Growth of L. bulgaricus under controlled pH (commonly used by the starter cultures production industry) results in cells more sensitive to stresses frequently encountered by the cells during starter cultures preparation/storage/utilization.     

Factors affecting production and activity of phospholipase C by Yersinia enterocotttica

The production of phospholipase C by Yersinia enterocolitica strain SG was optimum at 37†C at pH 6·5. No enzyme activity could be detected when the organism was grown at extreme pH values (pH > 8·5 or <5·0). The enzyme production was maximum when the organism was grown under static conditions in TSB medium. All solvents and salts inhibited the enzyme activity, whereas loss of activity was 95% in presence of methanol (20%) and 99% in presence of sodium azide (0·2 mol/l). The enzyme activity was increased twofold in the presence of cyste-ine and decreased by 98% in the presence of sodium perchlorate (0·2 mol/1).     

Relationship between lysostaphin endopeptidase production and cell wall composition in Staphylococcus staphylolyticus.

Mutants of Staphylococcus staphylolyticus incapable of producing an extracellular staphylolytic glycylglycine endopeptidase were isolated and found to have cells in the population susceptible to lysis by this enzyme, as did the wild-type organism under conditions in which the endopeptidase was not produced. These results suggest that cultures of this organism normally contain a heterogeneous population of cells with regard to cell wall composition and susceptibility to the enzyme. Production of the endopeptidase appears to act as a selective pressure which removes the susceptible cells in the population as the enzyme appears in the medium. A comparison of the peptidoglycan of the wild-type organism grown under conditions in which the endopeptidase was produced with that of this organism grown under nonproducing conditions and with those of endopeptidase-less mutants showed that in the presence of the endopeptidase the cell population had peptidoglycan with shorter peptide cross bridges and a greater percentage of serine in these cross bridges than was found in cells grown in the absence of the enzyme. The inability of the endopeptidase to hydrolyze glycylserine and serylglycine peptide bonds suggests that at least part of the resistance this organism has to the endopeptidase is due to relative amounts of serine found in the peptide cross bridges of some cells in the population.     

The location of the arginine-specific carboxypeptidase in the membrane of Mycoplasma salivarium and its physiological functions

A non-penetrating probe, 2,4,6-trinitrobenzenesulfonate, inhibited the activity of the carboxypeptidase purified from the cell membranes of Mycoplasma salivarium and the same enzymatic activity of intact Mycoplasma cells as well. Growth of the organism in medium containing benzoylglycyl-L-arginine resulted in a higher pH and higher turbidity than growth in the same medium without this supplement. It was concluded that the enzyme existed in the outer surface of the membrane of the cells and probably functioned to supply the organism with arginine as an energy source.     

D-泛解酸内酯水解酶产生菌的筛选及产酶条件研究

筛选到一株产D-泛解酸内酯水解酶的菌株,经鉴定为串珠镶孢霉菌（Fusarium monili-forme)SW-902。产酶条件研究表明,用甘油作碳源,蛋白胨作氮源,初始pH8.0,温度26℃,摇瓶培养3d,产酶量最高,在60L发酵罐中通风发酵45h,产菌丝体生物量7.18g干菌体/L,D-泛解酸内酯水解酶酶活力达到0.92IU/g干菌体。     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Selective enumeration of Lactobacillus casei in yoghurt-type fermented milks based on a 15°C incubation temperature

A procedure was developed to enumerate selectively Lactobacillus casei populations in yoghurt-type fermented milks that can also contain strains of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus and Bifidobacterium infantis. Commercial LBS agar was acidified to pH 5.4, and the plates were incubated at 15°C for 14 days under anaerobic conditions. Acidification prevented the development of streptococci, and incubation at 15°C limited the development of the lactobacilli and the bifidobacteria. L. casei formed colonies on HHD medium which were different from those obtained with L. bulgaricus. Counts of L. casei on HHD confirmed results obtained on LBS - pH 5.4 medium and incubated at 15°C. L. casei did not form colonies on M17, nor did L. acidophilus or L. bulgaricus.     

Hydrolysis of lactose by immobilized microorganisms.

Cells of Lactobacillus bulgaricus, Escherichia coli, and Kluyveromyces (Saccharomyces) lactis immobilized in polyacrylamide gel beads retained 27 to 61% of the beta-galactosidase activity of intact cells. Optimum temperature and pH and thermostability of these microbial beta-galactosidases were negligibly affected by the immobilization. Km values of beta-galactosidase in immobilized cells of L. bulgaricus, E. coli, and K. lactis toward lactose were 4.2, 5.4, and 30 mM, respectively. Neither inhibition nor activation of beta-galactosidase in immobilized L. bulgaricus and E. coli appeared in the presence of galactose, but remarkable inhibition by galactose was detected in the case of the enzyme of immobilized K. lactis. Glucose inhibited noncompetitively the activity of three species of immobilized microbial cells. These kinetic properties were almost the same as those of free beta-galactosidase extracted from individual microorganisms. The activity of immobilized K. lactis was fairly stable during repeated runs, but those of E. coli and L. bulgaricus decreased gradually. These immobilized microbial cells, when introduced into skim milk, demonstrated high activity for converting lactose to monosaccharides. The flavor of skim milk was hardly affected by treatment with these immobilized cells, although the degree of sweetness was raised considerably.     

Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus grown in a chemically defined medium

We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of Lactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricus strains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.     

beta-Glucosidase of Penicillium funiculosum. I. Purification

A strain of Penicillium funiculosum, isolated in this laboratory, produced in high yield both endo- and exo-glucanases and beta-glucosidases, which were suitable for the saccharification of cellulosic materials. The isolation of the beta-glucosidase of this organism, which differs from other beta-glucosidases of fungi in its substrate specificity, by preparative electrophoresis, is described in this article. The organism was grown on a lactose-casein medium and the culture filtrate concentrated by ammonium sulfate precipitation and dialysis. Electrophoresis was carried out on large slabs of polyacrylamide gel in an anodicrun in the presence of borate at pH 7. After elution of active fractions, a cathodic run was made at pH 6.0. Two precipitations with ammonium sulfate resulted in a homogeneous enzyme (specific activity 174 IU/mg). A second isozyme was also produced by P. funiculosum on cellulose-wheat bran medium. This isozyme was purified by electrophoresis at pH 7.0 in the absence of borate and was obtained free from other isozymes of beta-glucosidase and cellulases.     

Membrane-bound sugar alcohol dehydrogenase in acetic acid bacteria catalyzes L-ribulose formation and NAD-dependent ribitol dehydrogenase is independent of the oxidative fermentation

To identify the enzyme responsible for pentitol oxidation by acetic acid bacteria, two different ribitol oxidizing enzymes, one in the cytosolic fraction of NAD(P)-dependent and the other in the membrane fraction of NAD(P)-independent enzymes, were examined with respect to oxidative fermentation. The cytoplasmic NAD-dependent ribitol dehydrogenase (EC 1.1.1.56) was crystallized from Gluconobacter suboxydans IFO 12528 and found to be an enzyme having 100 kDa of molecular mass and 5 s as the sedimentation constant, composed of four identical subunits of 25 kDa. The enzyme catalyzed a shuttle reversible oxidoreduction between ribitol and D-ribulose in the presence of NAD and NADH, respectively. Xylitol and L-arabitol were well oxidized by the enzyme with reaction rates comparable to ribitol oxidation. D-Ribulose, L-ribulose, and L-xylulose were well reduced by the enzyme in the presence of NADH as cosubstrates. The optimum pH of pentitol oxidation was found at alkaline pH such as 9.5-10.5 and ketopentose reduction was found at pH 6.0. NAD-Dependent ribitol dehydrogenase seemed to be specific to oxidoreduction between pentitols and ketopentoses and D-sorbitol and D-mannitol were not oxidized by this enzyme. However, no D-ribulose accumulation was observed outside the cells during the growth of the organism on ribitol. L-Ribulose was accumulated in the culture medium instead, as the direct oxidation product catalyzed by a membrane-bound NAD(P)-independent ribitol dehydrogenase. Thus, the physiological role of NAD-dependent ribitol dehydrogenase was accounted to catalyze ribitol oxidation to D-ribulose in cytoplasm, taking D-ribulose to the pentose phosphate pathway after being phosphorylated. L-Ribulose outside the cells would be incorporated into the cytoplasm in several ways when need for carbon and energy sources made it necessary to use L-ribulose for their survival. From a series of simple experiments, membrane-bound sugar alcohol dehydrogenase was concluded to be the enzyme responsible for L-ribulose production in oxidative fermentation by acetic acid bacteria.     

Kluyveromyces bulgaricus yeast lectins. Isolation of N-acetylglucosamine and galactose-specific lectins: their relation with flocculation

Kluyveromyces bulgaricus is a yeast which, upon culture in a calcium-enriched glucose-peptone medium, flocculates. Its flocculation can be reversed by the addition of galactose. In this paper, it is shown that two lectins can be isolated either from the concentrated culture broth or from the supernatant of deflocculated cells suspended in galactose solution. The N-acetylglucosamine-specific lectin, at pH 7.4, agglutinates untreated sheep red blood cells, but agglutinates neither untreated rabbit red blood cells nor glutaraldehyde-fixed sheep or rabbit red blood cells. Conversely, at pH 4.5, this lectin agglutinates glutaraldehyde-fixed sheep red blood cells. The galactose-specific lectin, at pH 7.4, agglutinates both untreated and glutaraldehyde-fixed rabbit red blood cells but does not agglutinate untreated or glutaraldehyde-fixed sheep red blood cells. At pH 4.5, this lectin agglutinates both glutaraldehyde-fixed sheep and rabbit red blood cells and induces flocculation of deflocculated K. bulgaricus cells. In all cases, the agglutination and the flocculation induced by one of these two lectins were inhibited by free or conjugated N-acetyl-D-glucosamine or by free or conjugated D-galactose, respectively. No glycosylhydrolase activity could be detected in the purified lectins.     

Recovery of exocellular acid phosphatase activity on Saccharomyces mellis after treatment of the organism with reagents that affect the cell surface

Derepressed cells of Saccharomyces mellis were treated in one of several different ways to either elute or inactivate the exocellular enzyme, acid phosphatase. The enzyme was either (i) eluted from resting cells with 0.5 m KCl plus 0.1% beta-mercaptoethanol, (ii) eluted from exponential phase cells by growing the organism in derepressing media containing 0.5 m KCl, or (iii) inactivated on exponential phase cells by adding sufficient acid or base to growth media to destroy the enzyme but not enough to kill the cells. These treatments did not affect viability. Treated cells were transferred to fresh growth media or some other reaction mixture, and the kinetics of recovery of acid phosphatase activity was studied. In these reaction mixtures, enzyme was synthesized only by actively growing cells. Treated resting cells were indistinguishable from untreated, repressed resting cells in that the organism inoculated into complete growth medium remained in the lag phase for approximately 6 hr before both growth and enzyme synthesis began. Exponential phase derepressed cells treated by method (ii) or (iii) were transferred to fresh medium under conditions that allowed growth to continue. The cells immediately started to manufacture enzyme at a rate greater than normal until the steady-state level was reached, thus demonstrating a feedback control system. Exponential phase repressed cells were also transferred to fresh derepressing media under conditions which sustained growth. Though these cells began to grow immediately, there was a lag before acid phosphatase synthesis began followed by a lengthy inductive period. The length of the period of induction could be correlated with the polyphosphate content of the cells. As the supply of polyphosphate neared exhaustion, the rate of synthesis increased rapidly until it was greater than normal; this differential rate was sustained until the steady-state concentration was reached. When derepressed cells grow in a medium containing 0.5 m KCl, some acid phosphatase activity is found free in the culture fluid and some remains firmly attached to the cells despite the presence of the salt. The bound activity is subject to feedback control, but the steady-state level of this activity on the cells is only one-third that of the acid phosphatase on cells growing in nonsaline media. The extracellular phosphatase is produced at a rate that is several-fold greater than that of the exocellular enzyme in a nonsaline medium. The synthesis of the extracellular enzyme does not seem to be controlled by a feedback mechanism but is produced at a maximal rate as long as the cells are growing.     

Induction of tryptophan oxygenase by dexamethasone in isolated hepatocytes. Dependence on composition of medium and pH.

Hepatocytes were isolated from perfused rat livers. 4 x 10-6 cells/ml were incubated at at 37 degrees C in different media in the absence and presence of a steroid hormone, dexamethasone phosphate (2 x 10-5 M). 1. Hormonal enzyme induction occurred in cells suspended in a simple salt medium, devoid of amino acids and macromolecules. This induction was completely blocked by addition of either actinomycin D (2 mu-g/ml) or cycloheximide (50 mu-g/ml). 2. Incubation of cells in media containing defatted albumin did not enhance hormonal enzyme induction, although disintegration of cells during incubation was reduced. Addition of a crude albumin fraction reduced tryptophan oxygenase induction and dextran completely blocked enzyme induction by dexamethasone. 3. An increase of dexamethasone concentration in the presence of albumin to 9 x 10-5 M was unable to raise enzyme induction further, and a still higher concentration of hormone, 3 x 10-4 M, resulted in reduced enzyme induction. 4. The hormonal induction of tryptophan oxygenase was most pronounced when the pH of the medium was between 7.0 and 7.6, with an optium at 7.3. No induction was found when the pH of the medium was either 6.6 or 7.8. The basal tryptophan oxygenase activity was much less influenced by similar pH variations. It is concluded that hepatocytes in suspension are able to carry out hormone-stimulated enzyme synthesis and that factors influencing this process may be studied under controlled conditions in such systems.