scholarly journals Purification and properties of an aryl β-xylosidase from a cellulolytic extreme thermophile expressed in Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 645-650 ◽  
Author(s):  
R C Hudson ◽  
L R Schofield ◽  
T Coolbear ◽  
R M Daniel ◽  
H W Morgan
Keyword(s):  
Escherichia Coli ◽  
Thermal Inactivation ◽  
Competitive Inhibitor ◽  
Coli Strain ◽  
Extreme Thermophile ◽  
First Order ◽  
Optimal Activity ◽  
First Order Kinetics ◽  
Purification And Properties ◽  
Optimum Ph

An aryl beta-xylosidase was purified to homogeneity from an Escherichia coli strain containing a recombinant plasmid carrying a beta-xylosidase (EC 3.2.1.37) gene from the extremely thermophilic anaerobic bacterium isolate Tp8T6.3.3.1 (‘Caldocellum saccharolyticum’). It has a pI of 4.3 and shows optimal activity at pH 5.7. The enzyme is highly specific, acting on o- and p-nitrophenyl beta-D-xylopyranosides and minimally on p-nitrophenyl alpha-L-arabinopyranoside. It does not act on xylobiose. The Km for p-nitrophenyl beta-D-xylopyranoside at the optimum pH for activity is 10 mM, and at pH 7.0 is 6.7 mM. Xylose is a competitive inhibitor with Ki 40 mM. Thermal inactivation follows first-order kinetics at 65 and 70 degrees C with t1/2 values of 4.85 h and 40 min respectively. The t1/2 at 70 degrees C is increased 3-fold and 4-fold by the addition of 0.5 mg of BSA/ml and 2 mM-dithiothreitol respectively.

scholarly journals Purification and properties of a stable β-glucosidase from an extremely thermophilic anaerobic bacterium

1987 ◽  
Vol 243 (3) ◽  
pp. 779-787 ◽  
Author(s):  
M L Patchett ◽  
R M Daniel ◽  
H W Morgan
Keyword(s):  
Anaerobic Bacterium ◽  
Thermal Inactivation ◽  
First Order ◽  
First Order Kinetics ◽  
Purification And Properties ◽  
Wide Range ◽  
Optimum Ph ◽  
Exclusion Chromatography ◽  
Beta Glucosidase ◽  
Beta Galactosidase

A beta-glucosidase (EC 3.2.1.21) was purified to homogeneity from cell-free extracts of an extremely thermophilic anaerobic bacterium. The enzyme has an Mr of 43,000 as determined by molecular-exclusion chromatography, has a pI of 4.55 and shows optimum activity at pH 6.2. The enzyme is active against a wide range of aryl beta-glycosides and beta-linked disaccharides, with beta-galactosidase activity only slightly less than beta-glucosidase activity, and significant beta-xylosidase activity. Lineweaver-Burk plots for p-nitrophenyl beta-glucoside, o-nitrophenyl beta-glucoside and cellobiose substrates are biphasic concave-downwards. Inhibition of the beta-glucosidase by substrates and glucose is negligible. Thermal inactivation follows first-order kinetics, with t1/2 (65 degrees C) 45 h, t1/2 (75 degrees C) 47 min and t1/2 (85 degrees C) 1.4 min and a deactivation energy of 380 kJ/mol at pH 6.2. At pH 7.0, which is the optimum pH for thermostability, t1/2 (75 degrees C) is 130 min. At 75 degrees C, at pH 6.2, the thermostability is enhanced about 8-fold by 10% (w/v) glycerol, about 6-fold by 0.2 M-cellobiose and about 3-fold by 5 mM-dithiothreitol and 5 mM-2-mercaptoethanol.

Purification and properties of 6-phosphogluconate dehydrogenase from Penicillium duponti and Penicillium notatum

1972 ◽  
Vol 18 (8) ◽  
pp. 1289-1298 ◽  
Author(s):  
Hugh M. Miller ◽  
Maxwell G. Shepherd
Keyword(s):  
Protein Concentration ◽  
Thermal Inactivation ◽  
Sodium Dodecylsulfate ◽  
Thermophilic Enzyme ◽  
Penicillium Notatum ◽  
First Order ◽  
Phosphogluconate Dehydrogenase ◽  
First Order Kinetics ◽  
High Protein Concentration ◽  
Purification And Properties

6-Phosphogluconate dehydrogenase was isolated and partially purified from the thermophilic fungus Penicillium duponti and the mesophilic fungus Penicillium notatum. The specific activities of the purified enzymes were 17.5 and 22.0 respectively. Optimal activity was obtained at pH 8.0 for both enzymes. Non-linear Arrhenius plots were found for both enzymes with breaks at 30 °C for P. duponti 6-phosphogluconate dehydrogenase and 19 °C for P. notatum 6-phosphogluconate dehydrogenase. The thermal inactivation of 6-phosphogluconate dehydrogenase from both fungi exhibited first order kinetics, and the rate of inactivation for the thermophilic enzyme between 25 °C and 45 °C was only 25% that of the mesophilic enzyme. 6-Phosphogluconate dehydrogenase from both sources was protected from thermal inactivation by 6-phosphogluconic acid, high salt concentration, and high protein concentration. The thermophilic enzyme was found to be more resistant to the denaturants urea, acetamide, and sodium dodecylsulfate.

scholarly journals Suicidal inactivation and labelling of ammonia mono-oxygenase by acetylene

1985 ◽  
Vol 227 (3) ◽  
pp. 719-725 ◽  
Author(s):  
M R Hyman ◽  
P M Wood
Keyword(s):  
Protective Effect ◽  
Gel Electrophoresis ◽  
Ammonia Oxidation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Competitive Inhibitor ◽  
Ion Concentrations ◽  
First Order ◽  
First Order Kinetics ◽  
Labelling Experiment ◽  
Suicide Substrate

Acetylene brings about a progressive inactivation of ammonia mono-oxygenase, the ammonia-oxidizing enzyme in Nitrosomonas europaea. High NH4+ ion concentrations were protective. The inactivation followed first-order kinetics, with a rate constant of 1.5 min-1 at saturating concentrations of acetylene. If acetylene was added in the absence of O2, the cells remained active until O2 was re-introduced. A protective effect was also demonstrated with thiourea, a reversible non-competitive inhibitor of ammonia oxidation. Incubation of cells with [14C]acetylene was found to cause labelling of a single membrane polypeptide. This ran on dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr value of 28 000. It is concluded that acetylene is a suicide substrate for the mono-oxygenase. The labelling experiment provides the first identification of a constituent polypeptide of ammonia mono-oxygenase.

Effect of temperature and/or pressure on lactoperoxidase activity in bovine milk and acid whey

2001 ◽  
Vol 68 (4) ◽  
pp. 625-637 ◽  
Author(s):  
LINDA R. LUDIKHUYZE ◽  
WENDIE L. CLAEYS ◽  
MARC E. HENDRICKX
Keyword(s):  
High Temperature ◽  
Atmospheric Pressure ◽  
Thermal Inactivation ◽  
Bovine Milk ◽  
Antagonistic Effect ◽  
Effect Of Temperature ◽  
Pressure Treatment ◽  
First Order ◽  
First Order Kinetics ◽  
High Temperature Sensitivity

At atmospheric pressure, inactivation of lactoperoxidase (LPO) in milk and whey was studied in a temperature range of 69–73 °C and followed first order kinetics. Temperature dependence of the first order inactivation rate constants could be accurately described by the Arrhenius equation, with an activation energy of 635·3±70·7 kJ/mol for raw bovine milk and 736·9±40·9 kJ/mol for diluted whey, indicating a very high temperature sensitivity. On the other hand, LPO is very pressure resistant and not or only slightly affected by treatment at pressure up to 700 MPa combined with temperatures between 20 and 65 °C. Both for thermal and pressure treatment, stability of LPO was higher in milk than in diluted whey. Besides, a very pronounced antagonistic effect between high temperature and pressure was observed, i.e. at 73 °C, a temperature where thermal inactivation at atmospheric pressure occurs rapidly, application of pressure up to 700 MPa exerted a protective effect. At atmospheric pressure, LPO in diluted whey was optimally active at a temperature of about 50 °C. At all temperatures studied (20–60 °C), LPO remained active during pressure treatment up to 300 MPa, although the activity was significantly reduced at pressures higher than 100 MPa. The optimal temperature was found to shift to lower values (30–40 °C) with increasing pressure.

scholarly journals Purification and properties of cystathionine β-lyase from Arabidopsis thaliana overexpressed in Escherichia coli

1996 ◽  
Vol 320 (2) ◽  
pp. 383-392 ◽  
Author(s):  
Stéphane RAVANEL ◽  
Dominique JOB ◽  
Roland DOUCE
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Biochemical Properties ◽  
Coli Strain ◽  
High Yield ◽  
Purification And Properties ◽  
Key Enzyme ◽  
Schiff Base Formation ◽  
Insight Into ◽  
Base Formation

Cystathionine β-lyase is a key enzyme in sulphur metabolism that catalyses the second reaction specific for methionine biosynthesis, the pyridoxal 5´-phosphate-dependent β-cleavage of cystathionine to produce homocysteine. To obtain insight into the biochemical properties of the plant enzyme, the cDNA encoding cystathionine β-lyase from Arabidopsis thaliana was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated at high yield (29 mg of pure protein/litre of cell culture) using an efficient two-step purification procedure. Physicochemical properties of the Arabidopsis cystathionine β-lyase were similar to those previously reported for the bacterial enzymes. In particular, the native recombinant protein is a tetramer composed of four identical subunits of 46 kDa, each being associated with one molecule of pyridoxal 5´-phosphate. Interaction between the apoenzyme and pyridoxal 5´-phosphate is extremely tight, being characterized by a Kd value of 0.5 µM. Purification and sequencing of the phosphopyridoxyl peptide established that Schiff base formation between the cofactor and the holoenzyme occurs at lysine-278. The substrate specificity of the recombinant cystathionine β-lyase resembles that of the enzyme isolated from other sources, cystathionine and djenkolate being the most effective substrates. The cystathionine analogue aminoethoxyvinylglycine irreversibly inactivates the recombinant cystathionine β-lyase. The inactivation is accompanied by dramatic modification of the spectral properties of the enzyme that can be attributed to the attack of the azomethine linkage between pyridoxal 5´-phosphate and lysine-278 of the polypeptide by aminoethoxyvinylglycine.

scholarly journals The concerted inactivation of Escherichia coli uridine diphosphate galactose 4-epimerase by sugar nucleotide together with a free sugar

1976 ◽  
Vol 155 (2) ◽  
pp. 225-229 ◽  
Author(s):  
P Blackburn ◽  
W Ferdinand
Keyword(s):  
Escherichia Coli ◽  
Free Sugar ◽  
Uridine Diphosphate ◽  
Hydroxymethyl Group ◽  
Free Sugars ◽  
Sugar Nucleotides ◽  
Sugar Nucleotide ◽  
First Order ◽  
First Order Kinetics ◽  
Uridine Diphosphate Galactose

1. The combined effect of the sugar nucleotides UDP-D-fucose or UDP-D-glucuronic acid together with the free sugars D-fucose or L-arabinose is the inactivation of the Escherichia coli enzyme UDP-galactose 4-epimerase (EC 5.1.3.2). The sugar nucleotide or the free sugar alone or the sugar nucleotide plus 5'-Ump do not inactivate the enzyme. 2. The inactivation of the enzyme by its substrate UDP-D-glucose was not affected by the presence of free sugar. 3. In all cases the inactivation observed follows pseudo-first-order kinetics. 4. A comparison of various sugar nucleotides indicates that the hydroxymethyl group at position 6 of the sugar moiety of the natural substrates is important for substrate binding.

A New Kinetic Model for Thermal Inactivation of Microorganisms: Development and Validation Using Escherichia coli O157:H7 as a Test Organism†

2001 ◽  
Vol 64 (12) ◽  
pp. 2078-2082 ◽  
Author(s):  
LIHAN HUANG ◽  
VIJAY K. JUNEJA
Keyword(s):  
Escherichia Coli ◽  
Kinetic Model ◽  
Thermal Inactivation ◽  
Test Organism ◽  
Nonlinear Behavior ◽  
Ground Beef ◽  
Inactivation Kinetics ◽  
Escherichia Coli O157 ◽  
First Order ◽  
Test Microorganism

A new kinetic model has been proposed to simulate the nonlinear behavior of survivor curves frequently observed in thermal inactivation of microorganisms. This model incorporates a time component into the first-order inactivation kinetics and is capable of describing the linear, convex, and concave survivor curves. The model was validated using Escherichia coli O157:H7 as a test microorganism. Ground beef (93% lean) samples inoculated to 107 to 108 CFU/g of meat were subjected to immersion heating at 55, 57.5, 60, 62.5, and 65°C, respectively, in a water bath. All the survivor curves in this study showed upward concavity. Linear and nonlinear regressions were used to fit the survivor curves to the linear first-order inactivation kinetics and the proposed model. Analyses showed that the new kinetic model provides a much better estimate of the thermal inactivation behavior of E. coli O157:H7 in ground beef.

scholarly journals Quantitative analysis of proton-linked transport system. β-Galactoside exit in Escherichia coli

1980 ◽  
Vol 188 (2) ◽  
pp. 467-473 ◽  
Author(s):  
I R Booth ◽  
W A Hamilton
Keyword(s):  
Escherichia Coli ◽  
Quantitative Analysis ◽  
Transport System ◽  
Rate Constants ◽  
The Other ◽  
First Order ◽  
First Order Kinetics ◽  
Solute Accumulation ◽  
External Ph

The exit of lactose and thiomethyl-beta-D-galactoside from Escherichia coli ML308-225 has been studied to determine the role of carrier-dependent (zero-trans efflux) and carrier-independent (leak) processes. On the basis of its sensitivity to p-chloromercuribenzene sulphonate the exit of lactose was found to be almost wholly mediated by the carrier. Consistent with this conclusion was the finding that the rate of exit of this sugar was dependent on the external pH, being considerably slower at acid pH. On the other hand exit of thiomethyl-beta-D-galactoside was found to be composed of both carrier-dependent and carrier-independent processes. Both processes exhibited first-order kinetics with the rate constants for zero-trans efflux and leak being 0.137 min-1 and 0.079 min-1, respectively. The relevance of these findings for out earlier proposal for the methods of attenuation of solute accumulation is discussed [Booth, Mitchell & Hamilton (1979) Biochem. J. 182, 687–696].

scholarly journals STUDIES ON THE LACTASE OF ESCHERICHIA COLI

1941 ◽  
Vol 24 (3) ◽  
pp. 377-397 ◽  
Author(s):  
H. P. Knopfmacher ◽  
A. J. Salle
Keyword(s):  
Escherichia Coli ◽  
Sodium Sulfide ◽  
Potassium Cyanide ◽  
Order Reaction ◽  
E Coli ◽  
First Order ◽  
Optimal Activity ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Slight Inhibition

A "lactase solution" was prepared from Escherichia coli. The mechanism of its action has been studied and changes in the rate of hydrolysis under various conditions investigated. The hydrolysis of lactose by the enzyme approximates the course of reaction of the integrated Michaelis-Menten equation. One molecule of enzyme combines with one molecule of substrate. E. coli lactase is readily inactivated at pH 5.0, and its optimal activity at 36°C. is reached between pH 7.0 and pH 7.5. The optimal temperature for its action was found to be 46°C. when determinations were carried out after an incubation period of 30 minutes. Its inactivation by heat follows the course of a first order reaction, and the critical thermal increment between the temperatures of 45°C. and 53°C. was calculated to be 56,400 calories per mol. The enzyme is activated by potassium cyanide, sodium sulfide, and cysteine, and irreversibly inactivated by mercuric chloride, silver nitrate, and iodine. After inactivation with copper sulfate partial reactivation is possible, while the slight inhibition brought about by hydrogen peroxide is completely reversible. The possible structure of the active groups of E. coli lactase as compared with other enzymes has been discussed.

Escherichia coli Thermal Inactivation Relative to Physiological State

2009 ◽  
Vol 72 (2) ◽  
pp. 399-402 ◽  
Author(s):  
D. GLENN BLACK ◽  
FEDERICO HARTE ◽  
P. MICHAEL DAVIDSON
Keyword(s):  
Escherichia Coli ◽  
Goodness Of Fit ◽  
Thermal Inactivation ◽  
Physiological State ◽  
Weibull Model ◽  
Growth Conditions ◽  
Coli Strain ◽  
Microbial Inactivation ◽  
Microbial Cells ◽  
K 12

Studies have explored the use of various nonlinear regression techniques to better describe shoulder and/or tailing effects in survivor curves. Researchers have compiled and developed a number of diverse models for describing microbial inactivation and presented goodness of fit analysis to compare them. However, varying physiological states of microorganisms could affect the measured response in a particular population and add uncertainty to results from predictive models. The objective of this study was to determine if the shape and magnitude of the survivor curve are possibly the result of the physiological state, relative to growth conditions, of microbial cells at the time of heat exposure. Inactivation tests were performed using Escherichia coli strain K-12 in triplicate for three growth conditions: statically grown cells, chemostat-grown cells, and chemostat-grown cells with buffered (pH 6.5) feed media. Chemostat cells were significantly less heat resistant than the static or buffered chemostat cells at 58°C. Regression analysis was performed using the GInaFiT freeware tool for Microsoft Excel. A nonlinear Weibull model, capable of fitting tailing effects, was effective for describing both the static and buffered chemostat cells. The log-linear response best described inactivation of the nonbuffered chemostat cells. Results showed differences in the inactivation response of microbial cells depending on their physiological state. The use of any model should take into consideration the proper use of regression tools for accuracy and include a comprehensive understanding of the growth and inactivation conditions used to generate thermal inactivation data.

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