Effect of Growth Temperature on Hydrolytic and Esterifying Activities from Pseudomonas fragi CRDA 037 Grown on Whey

The production of hydrolytic and esterifying activities of Pseudomonas fragi CRDA 037 grown on a whey-based medium was investigated at different temperatures over time. The optimal temperature was found to be critical and different for the production of both activities. The highest hydrolytic activity was detected with bacteria cultivated at between 24°C (149.2 U/liter) and 27°C (133.8 U/liter), while the highest production of ethyl valerate (esterifying activity) was observed by using biomass grown at 15°C (0.75 U/liter). When the fermentation temperature was increased, the incubation time necessary to reach the maximal concentration of both activities was reduced. Studies of the thermostability of both activities showed that the hydrolytic activity was more stable than the esterifying activity at 15 and 24°C. Statistical analysis allowed the determination of the equations that predicted the production of hydrolytic and esterifying activities as a function of time and growth temperature. The optimal assay temperatures for the hydrolytic and esterifying activities were 37°C and 12 to 15°C, respectively.     

Lipase from Pseudomonas fragi CRDA 323: Partial purification,characterization and interesterification of butter fat

Several strains of Pseudomonas were screened for lipase production using the rhodamine agar diffusion test. On the basis of the diameter of the halo produced, P. fragi CRDA 323 and P. putida ATCC 795 were considered to be good and weak lipase producers respectively. P. fragi, cultured in a 2-1 fermenter, produced a maximal amount of lipase after 3–4 days of incubation at 27°C. The lipase extract of P. fragi was obtained by acidification of culture supernatant at pH 4.0 and partially purified with ammonium sulphate precipitation. The majority of lipase activity (42%) was located in fraction IV, precipitated at 20%–40% of saturation, with a 19-fold enzyme purification. The K _m and V _max values for the partially purified enzymatic extract (fraction IV) were 0.70 mg/ml and 0.97 × 10^–3 U/min respectively. Fraction IV, which showed and optimum activity at pH 8.5, was used for the interesterification of butter fat in a microemulsion free co-surfactant system containing Span 60 (sorbitol monostearate) and Tween 60 (polyoxyethylene sorbitan monostearate) in the ratio 48:52 (v/v). The results showed that the interesterification of butter fat resulted in a considerable decrease in long-chain saturated fatty acids (C12:0, C14:0 and C16:0) with a concomitant increase in C18:0 and C18:1 at the sn-2 position of selected triacylglycerols. In addition, the results demonstrated an increase in the fatty acids (C12:0, C14:0 and 16:0) among the 1 and 3 positions of the triacylglycerol molecules of modified buffer fat accompanied by a decrease in C18:0 and C18:1.     

Epoxide hydrolase-catalyzed resolution of ethyl 3-phenylglycidate using whole cells of Pseudomonas sp.

A Pseudomonas sp. was isolated with enantioselective epoxide hydrolase activity to ethyl 3-phenylglycidate. Cells grown on sucrose and suspended in 10% (v/v) dimethyl formamide as co-solvent produced (2R,3S) ethyl 3-phenylglycidate with 95% ee and 26% yield in 12 h from 0.2% (w/v) of the racemate.     

The role of xylonolactone in xylonic acid production by Pseudomonas fragi

Summary Xylonic acid was produced by Pseudomonas fragi ATCC 4973 on 15% xylose medium with a yield of 96% of the theoretical. In the middle of the fermentation, growth was inhibited due to formation of inhibitory xylono--lactone. The spontaneous, non-enzymatic hydrolysis of lactone accounted for only 19% of the hydrolysis demand, thus explaining the accumulation of xylonolactone in the broth. It appeared that high amounts of xylono--lactone induced xylonolactonase enzyme, which brought about the total hydrolysis of xylonolactone.     

Detection of extracellular proteinase of Pseudomonas fragi by enzyme-linked immunosorbent assay

A double-antibody-sandwich, enzyme-linked immunosorbent assay was developed to detect an extracellular proteinase produced by Pseudomonas fragi. The method was capable of detecting 4 g/ml of the proteinase in spiked samples of buffer and broth and 4.2 g/ml in a broth culture of the organism. The assay detected the presence of proteinase at bacterial densities of approximately 10⁴ cfu/ml, which develop after incubation for 15 h at 25°C in a broth medium. All assays could be completed within 7 h. This assay is of value in plotting proteolytic expression in relation to the growth cycle of Ps. fragi in broth culture and may be of value, with development, in other more complex milieux.     

Ester Production by Pseudomonas fragi IV. Demonstration of Esterase Activity

Assay of the esterase activity of sonically treated cell-free extracts, whole cell suspensions, and supernatant fluid of Pseudomonas fragi cultures with a differential respirometer revealed that the esterases were intracellular. Polyacrylamide-gel electrophoresis demonstrated six bands of esterase activity, which revealed substrate specificity differences. Band 1 exhibited slow mobility, bands 2, 3, and 4 moderate mobility, and bands 5 and 6 rapid mobility. Six bands were active with alpha-naphthyl acetate, four bands with alpha-naphthyl propionate, and 5 bands with alphanaphthyl butyrate. These esterases appeared to be more active with aromatic esters than with aliphatic esters.     

Biodegradation of methyl and ethyl acetates by immobilized Pseudomonas esterophilus cells

A biofilter was created based on polyvinylformal foam and Pseudomonas esterophilus strain VKM V-1436D cells, which utilize methyl and ethyl acetates as sources of carbon and energy. A complete conversion of methyl and ethyl acetate (2000 mg/l) under flow conditions was reached. Because carboxyl esterase does not exhibit specificity for these esters, no adaptation period was required for switching the biofilter between biodegradation of methyl acetate and ethyl acetate.     

Ester synthesis in benzene by polyethylene glycol-modified lipase from Pseudomonas fragi 22.39B

Lipase (EC 3.1.1.3) from Pseudomonas fragi 22.39B was modified with polyethylene glycol. The modified lipase was soluble in organic solvents such as benzene and chlorinated hydrocarbons, and catalyzed the synthesis of esters from fatty acids and alcohols in these solvents. The longer the chain length of fatty acid, the higher the ester synthesis activity. A similar specificity was not observed with other substrates like alcohol. Values of K_m and V_max were revealed by kinetic study on the ester synthesis reaction with the modified lipase in benzene. Fatty acids with branched carbon chain at the position neighboring the carboxyl group did not serve as substrates of ester synthesis.     

Biodegradation of methyl and ethyl acetates by immobilized Pseudomonas esterophilus cells

A biofilter based on polyvinylformal foam and Pseudomonas esterophilus strain VKM V-1436D cells, which utilize methyl and ethyl acetates as sources of carbon and energy, was created. A complete conversion of methyl and ethyl acetate (2000 mg/l) under flow conditions was reached. Because carboxyl esterase does not exhibit specificity for these esters, no adaptation period was required for switching the biofilter between biodegradation of methyl acetate and ethyl acetate.     

Ultrastructural localization of an extracellular protease in Pseudomonas fragi by using the peroxidase-antiperoxidase reaction

An extracellular protease, which previously has been found to correlate with the appearance of bleblike evaginations on the cell wall of Pseudomonas fragi ATCC 4973, was purified 38-fold by ammonium sulfate precipitation and Sephadex chromatography to yield a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antiserum raised against the purified enzyme had an enzyme-linked immunosorbent assay titer of 4 X 10(7). The peroxidase antiperoxidase method was used to localize the neutral protease in P. fragi at the ultrastructural level. Electron microscopy of cell sections of this organism revealed that high concentrations of positive immunoperoxidase reaction product were located near the cell wall, whereas control sections stained with preimmune or heterologous serum did not show similar deposits to be present. These results are consistent with the hypothesis that blebs appearing on P. fragi contain high concentrations of neutral protease.     

Effects of Homologous Bacteriophage on Growth of Pseudomonas fragi WY in Milk

Pseudomonas fragi strain WY and its homologous bacteriophage were added in varying concentrations to sterile skim milk which was stored at 7 C for 72 hr. When the initial concentration of the bacterial host was 100,000/ml, addition of as few as 10 plaque-forming units per ml of bacteriophage resulted in significantly lower counts in treated skim milk than in the controls which contained no phage. There was no significant effect, however, when the phage input was 1 in 10 ml and the bacterial count was 1,000 or 100,00/ml. No differences in bacterial counts occurred even when the phage concentration was 1,000/ml if the initial bacterial concentration was only 1,000/ml.     

Thermal Resistance of Pseudomonas fragi in Milk Containing Various Amounts of Fat

Similar populations of Pseudomonas fragi were grown at 25 C for 20 hr or at 7 C for 7 days in milk containing 0, 10, and 20% fat; they were then heated at 48, 50, and 52 C in milk containing 0, 10, and 20% fat. After inoculation, the heating medium contained 2.1 x 10(6) to 6.9 x 10(6) organisms per milliliter. The P. fragi cells grown in skim milk had greater thermal resistance (D(52) = 3.0 to 3.1) than those grown in milk containing fat (D(52) = 1.9 to 2.5). The organisms grown at 7 C for 7 days in milk containing 10% fat were more resistant (D(52) = 3.0) than those grown in the same medium at 25 C for 20 hr (D(52) = 2.0). The presence of 0 to 20% milk fat in the heating medium had no apparent effect on the thermal resistance.     

Improvement of the enzymatic synthesis of ethyl valerate by esterification reaction in a solvent system

The present study reports the improved enzymatic synthesis of ethyl valerate (green apple flavor) by esterification reaction of ethanol and valeric acid in heptane medium. Lipase from Thermomyces lanuginosus (TLL) was immobilized by physical adsorption on polyhydroxybutyrate (PHB) particles and used as a potential biocatalyst. The effect of certain parameters that influence the ester synthesis was evaluated by factorial design. The experimental conditions that maximized the synthesis of ethyl valerate were 30.5°C, 18% m/v of biocatalyst (TLL–PHB), absence of molecular sieves, agitation of 234?rpm, and 1,000?mM of each reactant (ethanol and valeric acid). Under these conditions, conversion percentage ≈92% after 105?min of reaction was observed. Soluble TLL was also used as biocatalyst and the highest conversion was of 82% after 120?min of reaction. Esterification reaction performed in a solvent-free system exhibited conversion of 13% after 45?min of reaction catalyzed by immobilized lipase, while the soluble lipase did not exhibit catalytic activity. The synthesis of the ester was confirmed by Fourier transform infrared spectroscopy and gas chromatography–mass spectrometry analyses. After six consecutive cycles of ethyl valerate synthesis, the prepared biocatalyst retained ≈86% of its original activity.     

Ultrastructural localization of an extracellular protease in Pseudomonas fragi by using the peroxidase-antiperoxidase reaction.

An extracellular protease, which previously has been found to correlate with the appearance of bleblike evaginations on the cell wall of Pseudomonas fragi ATCC 4973, was purified 38-fold by ammonium sulfate precipitation and Sephadex chromatography to yield a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyclonal rabbit antiserum raised against the purified enzyme had an enzyme-linked immunosorbent assay titer of 4 X 10(7). The peroxidase antiperoxidase method was used to localize the neutral protease in P. fragi at the ultrastructural level. Electron microscopy of cell sections of this organism revealed that high concentrations of positive immunoperoxidase reaction product were located near the cell wall, whereas control sections stained with preimmune or heterologous serum did not show similar deposits to be present. These results are consistent with the hypothesis that blebs appearing on P. fragi contain high concentrations of neutral protease.     

Observations by Electron Microscopy on Pig Muscle Inoculated and Incubated with Pseudomonas fragi

Myofibrils from pig muscle inoculated and incubated with Pseudomonas fragi showed an extremely disrupted appearance as compared to uninoculated controls. There was an almost complete absence of material in the H zone, marked disruption of the A band (probably myosin), and some loss of dense material from the Z line. These changes indicated that marked proteolysis had occurred. Bacteria observed in spoiled muscle tissue exhibited protrusions or blebs on the outer surface of the cell walls. The blebs appeared to form detached globules that migrated into the muscle mass. Bacteria grown in non-muscle-containing media did not produce blebs, which indicates the blebs were induced by growth on muscle tissue. The possibility that the blebs and globules may contain a proteolytic enzyme responsible for myofibrillar disruption is discussed.     

Effect of Different Temperature Upshifts on Protein Synthesis by the Psychrotrophic Bacterium Pseudomonas fragi

Pseudomonas fragi, a psychrotroph bacterium involved in meat product spoilage, was shifted either from 5° to 20°C or 30°C and from 28° to 34°C. The heat-shocked cells in the mid-log phase rapidly reached the characteristic growth rate of the postshock temperature. The patterns of synthesized proteins were compared by autoradiography of two-dimensional gel electrophoregrams. The rates of synthesis, after transfer of cells from 5° to 30°C, 5° to 20°C, and 28° to 34°C, changed for 30, 26, and 21 proteins respectively, of which 19, 17, and 12 were increased respectively. Thirteen proteins changed similarly for the three treatments, and two of the seven overexpressed proteins were immunologically related to the Escherichia coli DnaK and GroEL heat shock proteins. From the four low-molecular-mass proteins, belonging to the family of DNA-binding cold shock proteins (CSPs) such as CS7.4, the major E. coli CSP [15], the amounts of C7.0 and C8.0 decreased rapidly after the upshifts, whereas that of E7.0 and E8.0 increased greatly. Received: 22 November 1995 / Accepted: 22 December 1995     

Ester Production by Pseudomonas fragi. II. Factors Influencing Ester Levels in Milk Cultures

Cultures of Pseudomonas fragi were grown at 21 C in sterile homogenized milk and reconstituted skim milk media supplemented with ethyl alcohol. Quantitative determinations of ethyl butyrate and ethyl hexanoate by gas-liquid chromatography showed definite increases in the concentrations of the two esters produced in these media in comparison to media not supplemented with ethyl alcohol. Supplementation with butyric acid in addition to ethyl alcohol generally elevated the ethyl butyrate concentration and usually depressed the cell count slightly. Aeration of any of the media during growth tended to reduce the cell population slightly. A relationship between increase in cell number and increase in concentration of esters during the growth of the culture was observed. Media containing high concentrations of ethyl alcohol plus milk fat or low-molecular-weight fatty acids were conducive to the production of a fruity aroma by P. fragi.