Purification and thermostability of β-galactosidase (lactase) from an autolytic strain of Streptococcus salivarius subsp. thermophilus

1989 ◽  
Vol 56 (1) ◽  
pp. 117-127 ◽  
Author(s):  
Byeong-Seon Chang ◽  
Raymond R. Mahoney
Keyword(s):  
Activation Energy ◽  
Half Life ◽  
Reaction Order ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Enzyme Concentration ◽  
Lactose Hydrolysis ◽  
Streptococcus Salivarius ◽  
First Order

Summaryβ-Galactosidase from an autolytic strain of Streptococcus salivarius subsp. thermophilus was purified 109-fold to near homogeneity. The yield of purified enzyme was 41% and the specific activity was 592 0-nitrophenyl β-D-galactopyranoside U/mg at 37 °C. Two isozymes were present, but only one subunit was detected, having a mol. wt of 116000. Enzyme stability was 37–83 times greater in milk than in buffer in the range 60–65 °C. At 60 °C the half-life in milk was 146 min. Denaturation in buffer was first-order, but in milk the overall reaction order with respect to enzyme concentration was ˜ 0·5. The activation energy for denaturation was 453 kJ/mol in milk and 372 kJ/mol in buffer. In milk the activation energy for lactose hydrolysis was 35·1 kJ/mol.

Simultaneous optimization of activity and stability of xylose reductase from D. nepalensis NCYC 3413 using statistical experimental design

2020 ◽  
Vol 27 ◽  
Author(s):  
Shwethashree Malla ◽  
Sathyanarayana N. Gummadi
Keyword(s):  
Half Life ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Reductase Activity ◽  
Xylose Reductase ◽  
Life Time ◽  
Economic Feasibility ◽  
Simultaneous Optimization ◽  
Physical Parameters ◽  
Statistical Experimental Design

Background: Physical parameters like pH and temperature play a major role in the design of an industrial enzymatic process. Enzyme stability and activity are greatly influenced by these parameters; hence optimization and control of these parameters becomes a key point in determining the economic feasibility of the process. Objective: This study was taken up with the objective to optimize physical parameters for maximum stability and activity of xylose reductase from D. nepalensis NCYC 3413 through separate and simultaneous optimization studies and comparison thereof. Method: Effects of pH and temperature on the activity and stability of xylose reductase from Debaryomyces nepalensis NCYC 3413 were investigated by enzyme assays and independent variables were optimised using surface response methodology. Enzyme activity and stability were optimised separately and concurrently to decipher the appropriate conditions. Results: Optimized conditions of pH and temperature for xylose reductase activity were determined to be 7.1 and 27 ℃ respectively, with predicted responses of specific activity (72.3 U/mg) and half-life time (566 min). The experimental values (specific activity 50.2 U/mg, half-life time 818 min) were on par with predicted values indicating the significance of the model. Conclusion: Simultaneous optimization of xylose reductase activity and stability using statistical methods is effective as compared to optimisation of the parameters separately.

Use of purified lyophilized human lactate dehydrogenase isoenzyme 5 in a study of measuring lactate dehydrogenase activity.

1989 ◽  
Vol 35 (8) ◽  
pp. 1774-1776 ◽  
Author(s):  
D A Smith ◽  
G C Moses ◽  
A R Henderson
Keyword(s):  
Lactate Dehydrogenase ◽  
Half Life ◽  
Specific Activity ◽  
Lactate Dehydrogenase Activity ◽  
Bovine Albumin ◽  
First Order ◽  
First Order Kinetics ◽  
The Stability ◽  
Lactate Dehydrogenase Isoenzymes ◽  
Excellent Stability

Abstract We examined the stability of human lactate dehydrogenase (EC 1.1.1.27) isoenzyme 5--purified to a specific activity of about 400 kU/g--when lyophilized in a buffered, stabilized matrix of bovine albumin. This isoenzyme was prepared with a final activity of about 500 U/L and stored at -20, 4, 20, 37, and 56 degrees C for as long as six months. This isoenzyme decayed with approximate first-order kinetics, with an estimated half-life at -20 degrees C of about 475 years. Stability of reconstituted samples stored at 20 or 4 degrees C was poor, suggesting that the reconstituted material should be used without delay; material stored at -20 degrees C showed excellent stability for 15 days. We propose that such preparations might be further investigated as standards for use in electrophoresis of lactate dehydrogenase isoenzymes.

Factors affecting the thermostability of β-galactosidase (Streptococcus salivarius subsp. thermophilus) in milk: a quantitative study

1989 ◽  
Vol 56 (5) ◽  
pp. 785-792 ◽  
Author(s):  
Byeong-Seon Chang ◽  
Raymond R. Mahoney
Keyword(s):  
Phosphate Buffer ◽  
Enzyme Stability ◽  
Divalent Cations ◽  
Whey Proteins ◽  
Milk Proteins ◽  
Enzyme Concentration ◽  
Streptococcus Salivarius ◽  
Factors Affecting ◽  
Micellar Casein ◽  
Acting In Concert

SummaryEnhanced stability of β-galactosidase (Streptococcus salivarius subsp. thermophilus) in milk is due to several milk components acting in concert. Milk salts stabilized the enzyme 3-fold compared to phosphate buffer. K+ was a better stabilizer than Na+. Omission of divalent cations, especially Mg2+, caused a marked drop in stability. Lactose stabilized enzyme stored in the frozen state but destabilized enzyme stored unfrozen. Caseinate stabilized the enzyme 8-fold in phosphate buffer but it stabilized 144-fold in the presence of lactose. In the presence of milk proteins, lactose was 25 times as effective as galactose and 100 times as effective as glucose at promoting stability; sucrose slightly destabilized the enzyme. Stability rose with increasing lactose concentration but declined with increasing enzyme concentration. In milk, soluble casein was the primary stabilizer; whey proteins and peptides had much less effect. Micellar casein had no effect on stability.

Kinetics of Rubber-Modified Nylon 6

2014 ◽  
Vol 887-888 ◽  
pp. 951-954
Author(s):  
Hong Kai Zhao ◽  
Hong Li Wang
Keyword(s):  
Activation Energy ◽  
Reaction Order ◽  
Styrene Butadiene Rubber ◽  
Butadiene Rubber ◽  
Exponential Factor ◽  
First Order ◽  
Adiabatic Approach ◽  
Research Findings ◽  
Styrene Butadiene ◽  
Kinetics Of

Kinetic parameters are calculated based on the reactive temperature rise curve measured by adiabatic approach at the temperature of 145 to 160 °C with the catalytic system of NaOH and acyl caprolactam End-capped butadiene-acrylonitrile rubber (CHTBN) or styrene-butadiene rubber (CHTBS). The reaction order is first order, the activation energy is between 72.91−73.16 kJ∙mol−1 and the pre-exponential factor is between 3.22×1011− 3.38×1011 mol1−n∙s−1 in the system of CHTBN/NaOH. While in CHTBS/NaOH, the reaction order is between 1.23-1.34, the activation energy is between 85.55－86.88 kJ∙mol−1 and the pre-exponential factor is between 4.52×1011−5.0 9×1011 mol1−n∙s−1. The adiabatic reaction kinetic model of caprolactam anion was constructed based on the existing research findings, by which the polymerizing reaction is simulated. The coincidence between the simulation results and the experimental data revealed that the model is reasonable and correct.

The kinetics of the thermal decomposition of normal paraffin hydrocarbons - V. Order of reaction over extended ranges of pressure

1952 ◽  
Vol 214 (1116) ◽  
pp. 20-35 ◽  
Keyword(s):  
Activation Energy ◽  
Reaction Order ◽  
Initial Pressure ◽  
Important Change ◽  
Second Order ◽  
Relative Probability ◽  
First Order ◽  
Molecular Reaction ◽  
Single Transition ◽  
Kinetics Of

Previous papers have provided evidence that the decomposition of a paraffin reduced to its limiting rate by nitric oxide is a molecular reaction. This reaction has an unusual pressure dependence, which has now been studied over a wider range, namely, up to 1600 mm, and down to 0⋅1 mm. In the higher part of the pressure range the reaction order changes from nearly the second to the first with increase in the initial pressure, as now confirmed by extended measurements on n -butane and n -pentane. In the lower part of the range another transition from second order to first with rise of pressure can be found, and is here shown by measurements on ethane, propane, n -butane, n -pentane and n -hexane. Thus with higher normal paraffins the reaction order is the second at the lowest pressure, decreases to the first, increases again towards the second and finally returns to the first at the highest pressure. Where this behaviour is shown the activation energy is also a function of the pressure. The activation energy for ethane is not a function of the pressure and, correspondingly, there is here a single transition from the second order at the lowest pressure to the first order at about 400 mm and up to at least 1600 mm. Over the range of pressure where the composite behaviour is observed, mass spectrometer analysis of the butane products reveals no important change in the relative probability of rupture at the C 1-2 and C 2-3 linkages respectively. In the region of the lower pressures, however, some of the primarily formed ethane splits to give ethylene and hydrogen more rapidly than can be accounted for by the decomposition rate of ethane as directly determined for comparable conditions.

In-vitro Kinetic Hydrolysis Study of Metronidazole Derivatives with Carvacrol and Eugenol Using Validated RP-HPLC Method

2020 ◽  
Vol 16 ◽  
Author(s):  
M. Alarjah
Keyword(s):  
Half Life ◽  
Hplc Method ◽  
Hydrolysis Rate ◽  
First Order ◽  
First Order Kinetics ◽  
Rp Hplc ◽  
Pharmacokinetic Properties ◽  
Pseudo First Order ◽  
Kinetic Hydrolysis

Background: Prodrugs principle is widely used to improve the pharmacological and pharmacokinetic properties of some active drugs. Much effort was made to develop metronidazole prodrugs to enhance antibacterial activity and or to improve pharmacokinetic properties of the molecule or to lower the adverse effects of metronidazole. Objective: In this work, the pharmacokinetic properties of some of monoterpenes and eugenol pro metronidazole molecules that were developed earlier were evaluated in-vitro. The kinetic hydrolysis rate constants and half-life time estimation of the new metronidazole derivatives were calculated using the validated RP-HPLC method. Method: Chromatographic analysis was done using Zorbbax Eclipse eXtra Dense Bonding (XDB)-C18 column of dimensions (250 mm, 4.6 mm, 5 μm), at ambient column temperature. The mobile phase was a mixture of sodium dihydrogen phosphate buffer of pH 4.5 and methanol in gradient elution, at 1ml/min flow rate. The method was fully validated according to the International Council for Harmonization (ICH) guidelines. The hydrolysis process carried out in an acidic buffer pH 1.2 and in an alkaline buffer pH 7.4 in a thermostatic bath at 37ºC. Results: The results followed pseudo-first-order kinetics. All metronidazole prodrugs were stable in the acidic pH, while they were hydrolysed in the alkaline buffer within a few hours (6-8 hr). The rate constant and half-life values were calculated, and their values were found to be 0.082- 0.117 hr-1 and 5.9- 8.5 hr., respectively. Conclusion: The developed method was accurate, sensitive, and selective for the prodrugs. For most of the prodrugs, the hydrolysis followed pseudo-first-order kinetics; the method might be utilised to conduct an in-vivo study for the metronidazole derivatives with monoterpenes and eugenol.

Catalyst Selection for Hydrogenation of 1,2-Dihydroacenaphthylene

1998 ◽  
Vol 63 (11) ◽  
pp. 1945-1953 ◽  
Author(s):  
Jiří Hanika ◽  
Karel Sporka ◽  
Petr Macoun ◽  
Vladimír Kysilka
Keyword(s):  
Activation Energy ◽  
Nickel Catalyst ◽  
Palladium Catalyst ◽  
Active Component ◽  
Reaction Order ◽  
Nickel Catalysts ◽  
Practical Application ◽  
Repeated Use ◽  
Selection For ◽  
Catalyst Selection

The activity of ruthenium, palladium, and nickel catalysts for the hydrogenation of 1,2-dihydroacenaphthylene in cyclohexane solution was studied at temperatures up to 180 °C and pressures up to 8 MPa. The GC-MS technique was used to identify most of the perhydroacenaphthylene stereoisomers, whose fractions in the product were found dependent on the nature of the active component of the catalyst. The hydrogenation was fastest on the palladium catalyst (3% Pd/C). The nickel catalyst Ni-NiO/Al2O3, which is sufficiently active also after repeated use, can be recommended for practical application. The activation energy of 1,2-dihydroacenaphthylene hydrogenation using this catalyst is 17 kJ/mol, the reaction order with respect to hydrogen is unity.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

Posttranslational modifications of recombinant myotube-synthesized human factor IX

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 130-138 ◽  
Author(s):  
Valder R. Arruda ◽  
James N. Hagstrom ◽  
Jeffrey Deitch ◽  
Terry Heiman-Patterson ◽  
Rodney M. Camire ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Posttranslational Modifications ◽  
Half Life ◽  
Specific Activity ◽  
Factor Ix ◽  
Serine Phosphorylation ◽  
Hemophilia B ◽  
Tyrosine Sulfation ◽  
Aav Vector ◽  
Carbohydrate Analysis

Abstract Recent data demonstrate that the introduction into skeletal muscle of an adeno-associated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical γ-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and γ-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX.

Vulcanization of Elastomers. 23. The Chemical Kinetics of Vulcanization Reactions and The Physical Properties of The Vulcanizates. I

1960 ◽  
Vol 33 (2) ◽  
pp. 335-341
Author(s):  
Walter Scheele ◽  
Karl-Heinz Hillmer
Keyword(s):  
Activation Energy ◽  
Physical Properties ◽  
Chemical Kinetics ◽  
Kcal Mole ◽  
First Order ◽  
Equilibrium Swelling ◽  
Kinetics Of ◽  
Kinetics Of Vulcanization ◽  
Limiting Value ◽  
Good Agreement

Abstract As a complement to earlier investigations, and in order to examine more closely the connection between the chemical kinetics and the changes with vulcanization time of the physical properties in the case of vulcanization reactions, we used thiuram vulcanizations as an example, and concerned ourselves with the dependence of stress values (moduli) at different degrees of elongation and different vulcanization temperatures. We found: 1. Stress values attain a limiting value, dependent on the degree of elongation, but independent of the vulcanization temperature at constant elongation. 2. The rise in stress values with the vulcanization time is characterized by an initial delay, which, however, is practically nonexistent at higher temperatures. 3. The kinetics of the increase in stress values with vulcanization time are both qualitatively and quantitatively in accord with the dependence of the reciprocal equilibrium swelling on the vulcanization time; both processes, after a retardation, go according to the first order law and at the same rate. 4. From the temperature dependence of the rate constants of reciprocal equilibrium swelling, as well as of the increase in stress, an activation energy of 22 kcal/mole can be calculated, in good agreement with the activation energy of dithiocarbamate formation in thiuram vulcanizations.

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