Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36

A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0.1 M-phosphate buffer, pH 6.6, being completely denatured above 70 degrees C in 1 min. It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 degrees C (minimum at 55 degrees C), but stability was very high above 80 degrees C in this buffer. D-values were determined at 10 degrees C intervals in the range 70-150 degrees C in SMS from which a Z value of 31.9 degrees C and an Ea of 8.82 X 10(4) J mol-1 were calculated; the half-life at 150 degrees C was 9 s. Instability at 55 degrees C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups. The extent of inactivation experienced at 80 degrees C was inversely related to the rate of heating to 80 degrees C, i.e. length of time spent in the neighbourhood of 55 degrees C. Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 degrees C, presumably due to substrate binding. Attempts to stabilize the enzyme at 55 degrees C by addition of EDTA or by adjusting the reaction pH to 4.2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented. Unlike the proteinase from Ps. fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 degrees C from 80, 100 or 150 degrees C, but did regain autolytic activity on cooling to below 50 degrees C to an extent dependent on the duration of holding at the lower temperature. It is suggested that on heating to approximately 55 degrees C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 degrees C.     

Isolation and characterization of heat stable proteinases from Pseudomonas isolate AFT 21

Pseudomonas strain AFT 21 produced three heat stable extracellular proteinases in milk and nutrient broth at 7 or 21 degrees C, but the proportions depended on medium and cultivation temperature. The three proteinases were EDTA- and o-phenanthroline-sensitive metalloenzymes and were not inhibited by N-ethylmaleimide or phosphoramidon. Proteinases I and II showed maximum activity at pH 7-7.5 and proteinase III at pH 8.5. All three enzymes showed maximum activity at 45-47.5 degrees C, but had relatively high (19-27% of maximum) activity at 4 degrees C. They were unstable at 55 degrees C in phosphate buffer, pH 6.6, or synthetic milk ultrafiltrate (SMUF) containing 12 mmol Ca2+, but were stabilized by short preheating at 100 degrees C. They were extremely heat stable in both phosphate buffer and SMUF, pH 6.6, at 70-150 degrees C. Their D-values at 140 degrees C were 69, 54 and 80 s respectively. The Z-values for Pseudomonas AFT 21 proteinase III in phosphate buffer and SMUF were 29.7 and 30.3 degrees C respectively; the corresponding activation energies for inactivation were 8.7 x 10(4) J mol-1 and 9.2 X 10(4) J mol-1.     

Growth of an extracellular proteinase-deficient strain of Pseudomonas fluorescens on milk and milk proteins

An extracellular proteinase-and lipase-deficient mutant of a psychrotroph, Pseudomonas fluorescens strain 32A, has been isolated and the absence of the proteinase enzyme confirmed by growth on differential media, enzyme assay and polyacrylamide gel electrophoresis. Competition between the parent and the mutant was observed when equal numbers of the 2 strains were inoculated together into raw skim-milk at 6 degrees C. Bitterness was detected at 6 degrees C in pasteurized skim-milk inoculated with the parent cells concurrent with the detection of proteolytic activity. In the case of the mutant, slight bitterness which did not increase with increasing cell numbers was detected in the absence of proteolysis. Mutant cells failed to grow on Na caseinate as the sole source of carbon. It was concluded that the extracellular proteinase, while not essential for growth in milk, does provide a selective advantage to the producer organism. This enzyme is, however, essential for growth on milk proteins and contributes to the development of bitterness in pasteurized milk.     

Determination of the extracellular lipases of Pseudomonas fluorescens spp. in skim milk with the beta-naphthyl caprylate assay

A method based on the hydrolysis of beta-naphthyl caprylate (beta-NC) has been developed for quantitating extracellular lipase from Pseudomonas fluorescens. The assay was extremely sensitive to skim milk (SM); as little as 0.02 ml raw SM in a 2.0 ml reaction mixture resulted in an apparent loss of 50% of the lipase activity. Activity improved 3-fold when trypsin (50 micrograms/ml) was included in the reaction mixture. When super-simplex optimization was used to determine the optimum levels of beta-NC, Na taurocholate (NaTC), SM/lipase mixture and trypsin for maximum activity, NaTC was found to be unnecessary for activity. Subsequent addition of 15 mM-NaTC resulted in 80% loss of activity. On the other hand, NaTC was required for native lipase activity in the presence of SM. Native lipase was completely inhibited by heating at 70 degrees C for 2 min, while B52 lipase retained 75% of its activity under the same conditions. The assay was able to detect lipase produced by Ps. fluorescens B52 in SM at 5 degrees C when the cell density exceeded 10(8) colony forming units/ml. The presence of butterfat (3.5%) in the SM assay inhibited B52 lipase by 97%. The beta-NC assay gave results comparable to the tributyrin agar diffusion assay using cell-free extracts of ten strains of common dairy psychrotrophs. The results suggest that the beta-NC assay may be useful for determining lipase activity in raw SM.     

Purification and characterization of the extracellular proteinase of Pseudomonas fluorescens NCDO 2085

Pseudomonas fluorescens NCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 degrees C and pH 7.0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3.5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5.40 +/- 0.05 and a mol. wt of 40 200 +/- 2100. It is heat-stable having D-values at 74 and 140 degrees C of 1.6 and 1.0 min respectively; 40 and 70% of the original activity remained after HTST (74 degrees C/17 s) and ultra high temperature (140 degrees C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from other Pseudomonas spp.     

Peptidase profiles of Pseudomonas fluorescens: identification and properties.

The cell-associated peptidase profiles of 12 strains of Pseudomonas fluorescens (ATCC 948 and 11 related biotypes) were examined. Employing Analytab system API ZYM, a general, strong peptidase activity was detected using L-lysyl-, L-pyrrolidonyl-, L-arginyl-, L-alanyl-, and glycyl-glycyl-beta-naphthylamides as substrates. Conversely, L-tyrosyl-, L-phenylalanyl-, L-histidyl-, L-prolyl-, gamma-L-glutamyl-beta-naphthylamides substrates were hydrolyzed by only a few strains. The peptidases were active, therefore, on substrates responsible for the bitter taste in dairy products. Properties of hydrolytic systems showed no significant changes in the enzymatic profiles when cells were grown on different fermentation media. Enzyme activity was relatively stable during refrigerated (5 degrees C) and frozen (-18 degrees C) storage. The peptidases of P. fluorescens ATCC 948, considered as reference, and strain 22 were identified on Pro-beta-naphthylamides by Michaelis constant values of .528 and .394 mM, respectively, and by different optimal pH and temperature activity on Leu- and Pro-beta-naphthylamides. The peptidase activity on Gly-Phe-beta-naphthylamide in P. fluorescens 30 had optimal values at pH 7.50 and 45 degrees C. These results confirm the relations defined in the enzymatic identification phase and suggest the presence of any analogous peptidases in the biovars of P. fluorescens considered.     

荧光假单胞菌噬菌体KS461-2的分离及其生物学特性研究

从2-酮基-D-葡萄糖酸产生菌荧光假单胞菌A46异常发酵液中分离得到1种噬菌体,将其命名为KS461-2。电镜观察表明,噬菌体KS461-2呈蝌蚪状。根据ICTV对病毒的分类标准,噬菌体KS461-2属长尾噬菌体科,为烈性噬菌体。噬菌体KS461-2对宿主菌的最佳感染复数为0．01,温度和pH对其稳定性具有显著的影响。一步生长曲线显示,噬菌体KS461-2的潜伏期约30min,裂解期约135min,裂解量为24。SDS-PAGE显示,噬菌体KS461-2有5种主要的结构蛋白,其分子量分别为66．0、61．0、42．5、34．7和14．4kDa。     

Bioinformatic characterization of the extracellular lipases from Kluyveromyces marxianus

Lipases are hydrolytic enzymes that break the ester bonds of triglycerides, generating free fatty acids and glycerol. Extracellular lipase activity has been reported for the nonconventional yeast Kluyveromyces marxianus, grown in olive oil as a substrate, and the presence of at least eight putative lipases has been detected in its genome. However, to date, there is no experimental evidence on the physiological role of the putative lipases nor their structural and catalytic properties. In this study, a bioinformatic analysis of the genes of the putative lipases from K. marxianus L-2029 was performed, particularly identifying and characterizing the extracellular expected enzymes, due to their biotechnological relevance. The amino acid sequence of 10 putative lipases, obtained by in silico translation, ranged between 389 and 773 amino acids. Two of the analysed putative proteins showed a signal peptide, 25 and 33 amino acids long for KmYJR107Wp and KmLIP3p, and a molecular weight of 44.53 and 58.23 kDa, respectively. The amino acid alignment of KmLIP3p and KmYJR107Wp with the crystallized lipases from a patatin and the YlLip2 lipase from Yarrowia lipolytica, respectively, revealed the presence of the hydrolase characteristic motifs. From the 3D models of putative extracellular K. marxianus L-2029 lipases, the conserved pentapeptide of each was determined, being GTSMG for KmLIP3p and GHSLG for KmYJR107Wp; besides, the genes of these two enzymes (LIP3 and YJR107W) are apparently regulated by oleate response elements. The phylogenetic analysis of all K. marxianus lipases revealed evolutionary affinities with lipases from abH15.03, abH23.01, and abH23.02 families.     

Autolysis of the proteinase from Pseudomonas fluorescens.

The gene encoding the proteinase from Pseudomonas fluorescens was cloned and sequenced in an effort to identify the cleavage sites involved in its autolysis at 50 degrees C. A single open reading frame consisting of 1449 nucleotides, encoding a protein of 482 amino acids, was found. Analysis of the N-terminal amino acid sequence of the purified proteinase indicated the presence of a prosequence consisting of 13 amino acid residues. The molecular weight of the mature protein was calculated as 48,900 based on the deduced amino acid sequence, which was consistent with that of the purified proteinase as determined by sodium dodecylsulfate-PAGE. Greater than 90% loss of proteolytic activity was observed upon heating at 50 degrees C for 2 min compared with the unheated enzyme. Incubation of the proteinase at 50 degrees C led to disappearance of the intact enzyme, as shown by sodium dodecyl sulfate-PAGE, whereas it was stable in the presence of the protease inhibitor o-phenanthroline. Autolytic fragments were fractionated by reverse-phase HPLC and subjected to N-terminal amino acid sequence analysis in an effort to determine the cleavage sites. The cleavage profile was not definitive; however, amino acid residues with small side chain groups, such as glycine or alanine, were frequently found adjacent to the cleavage sites.     

Effect of carbon dioxide on growth and extracellular enzyme production by Pseudomonas fluorescens B52

The effect of carbon dioxide (30 mM.l-1) on growth and extracellular protease and lipase production by Pseudomonas fluorescens B52 growing in a simulated milk medium at 7 degrees C in fermenter was investigated. The addition of carbon dioxide was shown to have a differential effect with extracellular enzyme production, particularly lipase, being inhibited to a greater extent than bacterial growth and final cell numbers.     

Peptidases from two strains of Pseudomonas fluorescens: partial purification, properties and action on milk

Pseudomonas fluorescens strains 240 and 32A expressed cell-associated peptidase activity which was shown by subcellular fractionation to be primarily intracellular. Two peptidases were partly purified from strain 32A. One specifically hydrolysed N-alpha-benzoyl-DL-arginine-4-nitroanilide and was termed endopeptidase and the other hydrolysed L-lysine- and L-leucine-4-nitroanilide and was termed aminopeptidase. The endopeptidase had very low activity on bovine serum albumin compared with that of trypsin and probably was not a proteinase. The endopeptidase had a mol. wt of 33,000 and a pH optimum of 8.0. The enzyme was stimulated by Ca2+ and Mg2+ and inhibited by Co2+, Mn2+, Hg2+, Zn2+ and leupeptin. Soya bean trypsin inhibitor and phenylmethane sulphonyl fluoride (PMSF) had no effect on its activity. The aminopeptidase had a mol. wt of 44,000 and a pH optimum of 8.0. It was inhibited by all the metal ions mentioned above and by PMSF. Little proteolysis was found when ultra high temperature (UHT) sterilized milk was treated with cell-free extract from strain 32A. It was concluded that the cell-associated peptidases from Pseudomonas strains normally present in raw milk may not contribute significantly to the deterioration of UHT sterilized milk.     

Physiology and behavior of Pseudomonas fluorescens single and dual strain biofilms under diverse hydrodynamics stresses

Three selected Pseudomonas fluorescens strains (the type strain and two strains originally isolated from a dairy processing plant - D3-348 and D3-350) were used to form turbulent and laminar flow-generated biofilms under laboratorial conditions using flow cell reactors with stainless steel substrata. The D3-348 and D3-350 strains were also used to form dual biofilms. Biofilm phenotypic characteristics, such as respiratory activity, total and culturable cells, biomass, total and matrix proteins and polysaccharides were compared. Biofilm mechanical stability, as a major feature involved in biofilm persistence, was also assessed using a rotating device system. The results indicate that hydrodynamic conditions have a remarkable impact on biofilm phenotype. Turbulent biofilms were more active, had more mass per adhesion surface area, a higher number of total and culturable cells, a higher amount of total proteins per gram of biofilm, similar matrix proteins and identical (D3-348 and D3-350 single and dual biofilms) or smaller (type strain) total and matrix polysaccharides content than their laminar counterparts. Biofilms formed by the type strain revealed a considerable higher amount of total and culturable cells and a higher amount of total proteins (turbulent biofilms) and total and matrix polysaccharides per gram of biofilm than single and dual biofilms formed by the other strains. Mechanical stability assays disclosed that biofilms formed by both type and D3-348 strains had the highest resistance to removal when exposed to mechanical stress. Dual strain biofilms population analysis revealed an apparent co-existence, evidencing neutral interactions. The overall results provided useful information regarding a broad spectrum of P. fluorescens biofilm phenotypic parameters, which can contribute to control and model biofilm processes in food industry.     

Purification and characterization of three extracellular proteinases produced by Pseudomonas fluorescens INIA 745, an isolate from ewe's milk.

Three proteinases were isolated from culture medium of Pseudomonas fluorescens INIA 745 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, and Sephadex G-100 chromatography. Optimal temperature for enzymatic activity was 45 degrees C for all three proteinases. The pH optimum of proteinases I and II was found to be 7.0, while that of proteinase III was 8.0. Divalent metal ions like Cu2+, Co2+, Zn2+, Fe2+, and Hg2+ were inhibitory to proteinase activity while Ca2+, Mg2+, and Mn2+ had little or no inhibitory effect. The three enzymes were strongly inhibited by EDTA and 1,10-phenantroline and partially by cysteine. The three enzymes are metalloproteinases since they were inhibited by chelators and reactivated by Co2+, Mn2+, Cu2+, and Zn2+. The Km values of proteinases I, II, and III for casein were calculated to be 3.2, 2.6, and 5.2 mg/ml, respectively. Proteinases II and III rapidly degraded beta-casein, with preference to alphas1-casein, whereas proteinase I hydrolyzed both casein fractions at a slow rate.     

Purification and characterization of an extracellular protease produced by Pseudomonas fluorescens M3/6.

Pseudomonas fluorescens strain M3/6 was inoculated into reconstituted NDM and incubated at 7 degrees C for 46 d. A significant amount of extracellular protease was produced, mainly during the latter part of the culture's life cycle. The protease was purified using ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The isolated protease had activity on azocasein, alpha-, beta-, and kappa-caseins and a plasmin substrate but did not have plasminogen activator activity. The protease had a molecular weight of 45 kDa, an isoelectric point of pH 8.25, a broad temperature and pH range for activity, and was less heat stable in the isolated form than in the cell-free extract.     

Purification and characterization of the lipase from Pseudomonas fluorescens HU380

A lipase, which markedly splits polyunsaturated fatty acid ester (PUFA) bonds, from newly isolated Pseudomonas fluorescens HU380 was purified. The purification procedure included Phenyl-Toyopearl fractionation, DEAE-Sepharose chromatography, and Superdex-200HR chromatography. The enzyme was purified 24.3-fold with a yield of 14% and a specific activity of 9854 U/mg. Its molecular weight was estimated on SDS-PAGE to be 64,000. The optimum pH and temperature were 8.5 and 45 degrees C, respectively. The lipase was stable over the pH range of 6.0-7.0 at 30 degrees C for 24 h, and up to 40 degrees C at pH 7.0 for 60 min, when 0.1% Triton X-100 was present. The lipase preferably acted on short to middle-chain fatty acid simple methyl-esters and triglycerides, and cleaved mainly 1,3-ester bonds and to a lesser extent the 2-position ester bond of triolein. The lipase was inhibited by Co2+, Ni2+, Fe3+, Fe2+, and EDTA, and activated by Ca2+. Its N-terminal amino acid sequence was determined to be GVYDYKNFGTADSKALFSDAMAITLY, which exhibited considerable similarity with those of the lipases from other P. fluorescens strains, but no significant homology with other lipases. This lipase was able to decompose fats and oils that contained eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) without significantly affecting the contents of these fatty acids. The results suggest that the lipase may be useful when applied to the processing of industrial fats and oils containing EPA and DHA, such as fish oil splitting.     

产脂肪酶极端丝状真菌的筛选及其酶学特性

从南非德班市及其周边地区采集的76个土样中筛选得到99株嗜碱丝状真菌和51株嗜热丝状真菌,利用橄榄油乳化法以及对硝基苯酚法筛选得到三株具有底物专一性的菌株F4-173、F4-262、F8-67,经ITS分子生物学鉴定方法分别鉴定为Aspergillus fumigatus、Penicillium verrucosum、Penicillium chrysogenum。其中,Aspergillus fumigates F4-173可专一性分解菜籽油,其最适作用条件40℃、p H8.0,Penicillium verrucosum F4-262可专一性分解对硝基苯酚月桂酸酯,其最适作用条件为40℃、p H8.0,Penicillium chrysogenum F8-67可专一性分解大豆油,其酶液最适作用条件为50℃、p H6.5。      

Isolation and some properties of plastochromanol-8

Conditions suitable for preparative isolation, quantitative separation and determination of plastochromanol-8 (PC-8), and their mild oxidation products were elaborated. Mild oxidation of PC-8 alone and with gammatocopherol (gamma-T) equimolare mixture by benzoquinone gives four and two new products, respectively. These products give Emmerie-Engel reaction. Four of gamma-T, three of PC-8 and two mixed mild oxidation products were found in linseed (Lea number = 49). It was also found that PC-8 derived from plant oils was a natural antioxidant better inhibiting autoxidation than alpha-tocopherol in the model system in which lard was the substrate.