Kinetic laws for solid-supported enzymes

1970 ◽  
Vol 48 (10) ◽  
pp. 1498-1504 ◽  
Author(s):  
P. V. Sundaram ◽  
A. Tweedale ◽  
K. J. Laidler
Keyword(s):  
Substrate Concentration ◽  
Free Solution ◽  
Kinetic Behavior ◽  
Michaelis Constant ◽  
Solid Supports ◽  
High Substrate ◽  
Substrate Concentrations

Enzymes behave differently when attached to solid supports for four main reasons: (1) their conformations when they are supported may differ from those in free solution, (2) they act upon substrates in a different environment, (3) there will be partitioning of substrate between the support and the free solution, and (4) there will be effects due to diffusion of the substrate in the support. The present paper examines effects (3) and (4) and shows how rates will vary with substrate concentration. If factors (1) and (2) do not enter, rates in the limit of high substrate concentrations will be the same for the supported enzyme as in free solution. At low substrate concentrations, rates will be less for the supported enzyme if the substrate is less soluble in the support than in free solution, and the apparent Michaelis constant, Km(app.), will be greater; conversely, for higher solubility in the support, rates will be greater and Km(app.) smaller. Effect (4) leads to lower rates and higher Km(app.) values, except in the limit of high substrate concentrations. At a sufficiently low thickness of the support, depending upon the activity of the enzyme, the kinetic behavior becomes identical with that in free solution.

scholarly journals The catalase–hydrogen peroxide system. Kinetics of catalatic action at high substrate concentrations

1968 ◽  
Vol 110 (4) ◽  
pp. 617-620 ◽  
Author(s):  
Peter Jones ◽  
A. Suggett
Keyword(s):  
Hydrogen Peroxide ◽  
Reaction Time ◽  
Time Effect ◽  
Time Dependent ◽  
Inhibitory Effects ◽  
Michaelis Constant ◽  
High Substrate ◽  
Compound Ii ◽  
Kinetics Of ◽  
Substrate Concentrations

1. A re-examination of the catalase–hydrogen peroxide reaction at high substrate concentrations, by using the quenched-flow technique, reveals a more complex kinetic behaviour than that previously reported. At constant reaction time the catalatic process obeys Michaelis–Menten kinetics, but the apparent Michaelis constant is markedly time-dependent, whereas the conventional catalase activity is independent of time. 2. The kinetics of the ‘time effect’ were analysed and it is suggested that the effect derives from the formation of an inactive species (thought to be catalase Compound II). The process shows Michaelis–Menten kinetics, with a Michaelis constant equal to that for the catalatic reaction in the limit of zero reaction time. 3. It has been confirmed that certain buffer components have marked inhibitory effects on the catalatic reaction and that, in unbuffered systems, catalatic activity is substantially independent of pH in the range 4·7–10·5.

scholarly journals Enzymatic hydrolysis of extruded soybean meal at high substrate concentrations

2016 ◽  
pp. 63-73
Author(s):  
Anton Sharikov ◽  
Anna Sereda ◽  
Elena Kostyleva ◽  
Irina Velikoretskaya ◽  
Victor Polyakov
Keyword(s):  
Enzymatic Hydrolysis ◽  
Soybean Meal ◽  
Substrate Concentration ◽  
Degree Of Hydrolysis ◽  
High Substrate ◽  
Bacterial Protease ◽  
Biotechnological Processes ◽  
Enzyme Dosage ◽  
Hydrolysis Of ◽  
Substrate Concentrations

Extrusion as a pretreatment before enzymatic hydrolysis of soybean meal is an effective technique to eliminate antinutritional properties of the main thermostable soy proteins glycinin and ?-conglycinin for production of feed ingredients with enhanced properties. In terms of economic efficiency, biotechnological processes are preferable to carry out at high substrate concentrations. The aim of the investigation was to evaluate the influence of high substrate concentrations in the range of 26-32% and enzyme dosages (0.4 - 3.1 PU/g) on efficiency of hydrolysis of extruded toasted soybean meal with bacterial protease. The results showed that maximum degree of hydrolysis was 42.1% at the enzyme dosage of 3.6 PU/g and at the substrate concentration of 29.0%. The increase in the substrate concentration had a strong effect on the deterioration of dynamic viscosity of the hydrolysates from 0.2 to 5.82 Pa?s. A combination of extrusion cooking at 120?C and enzymatic treatment with ?Protolad B? protease enabled hydrolysis of glycinin and ?-conglycinin to peptides with molecular mass below 15 kDa.

scholarly journals Increased Selectivity of Novozym 435 in the Asymmetric Hydrolysis of a Substrate with High Hydrophobicity Through the Use of Deep Eutectic Solvents and High Substrate Concentrations

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 792 ◽  
Author(s):  
Yerko Fredes ◽  
Lesly Chamorro ◽  
Zaida Cabrera
Keyword(s):  
Substrate Concentration ◽  
Phosphate Buffer ◽  
Choline Chloride ◽  
Reaction Medium ◽  
Deep Eutectic Solvents ◽  
Novozym 435 ◽  
Chiral Drugs ◽  
High Substrate ◽  
Hydrolysis Of ◽  
Substrate Concentrations

The effects of the reaction medium and substrate concentration were studied on the selectivity of Novozym 435 using the asymmetric hydrolysis of dimethyl-3-phenylglutarate as a model reaction. Results show that the use of choline chloride ChCl:urea/phosphate buffer 50% (v/v) as a reaction medium increased the selectivity of Novozym 435 by 16% (e.e = 88%) with respect to the one in 100% phosphate buffer (e.e = 76%). Best results were obtained when high substrate concentrations (well above the solubility limit, 27-fold) and ChCl:urea/phosphate buffer 50% (v/v) as reaction medium at pH 7 and 30 °C were used. Under such conditions, the R-monoester was produced with an enantiomeric purity of 99%. Novozym 435 was more stable in ChCl:urea/phosphate buffer 50% (v/v) than in phosphate buffer, retaining a 50% of its initial activity after 27 h of incubation at pH 7 and 40 °C. Results suggest that the use of deep eutectic solvents (ChCl:urea/phosphate buffer) in an heterogeneous reaction system (high substrate concentration) is a viable and promising strategy for the synthesis of chiral drugs from highly hydrophobic substrates.

Multiplication of a Klebsiella pneumoniae Strain in Water at Low Concentrations of Substrates

1988 ◽  
Vol 20 (11-12) ◽  
pp. 117-123 ◽  
Author(s):  
D. van der Kooij ◽  
W. A. M. Hijnen
Keyword(s):  
Amino Acids ◽  
Water Treatment ◽  
Klebsiella Pneumoniae ◽  
Substrate Concentration ◽  
Treatment System ◽  
Water Treatment System ◽  
Low Concentrations ◽  
Ks Values ◽  
Substrate Concentrations

A K.pneumoniae strain, isolated from a water treatment system, was tested in growth measurements for its ability to multiply at substrate concentrations of a few micrograms per liter. The organism multiplied on mixtures of carbohydrates and amino acids at a substrate concentration of 1 µg of C of each compound per liter. Tests with individual compounds revealed that especially carbohydrates were utilized at low concentrations. The Ks values obtained for maltose and maltopentaose were 53 µg of C/l and 114 µg of C per liter, respectively. The significance of the growth of K.pneumoniae at low substrate concentrations is discussed.

scholarly journals The determination of binding parameters when the total and free substrate concentrations are not approximately equal

1979 ◽  
Vol 179 (3) ◽  
pp. 697-700 ◽  
Author(s):  
N Gains
Keyword(s):  
Substrate Concentration ◽  
Graphical Method ◽  
Enzyme Concentration ◽  
Binding Parameters ◽  
Free Concentration ◽  
Equilibrium Binding ◽  
Substrate Concentrations

By using a standard graphical method values of Km and V may be found that are independent of the conditions and assumptions that the total substrate concentration approximates to its free concentration and that Km is much larger than the enzyme concentration. The procedure is also applicable to the determination of equilibrium binding parameters of a ligand to a macromolecule.

scholarly journals The apparent Km is a misleading kinetic indicator

1986 ◽  
Vol 239 (1) ◽  
pp. 175-178 ◽  
Author(s):  
I W Plesner
Keyword(s):  
Substrate Concentration ◽  
Kinetic Data ◽  
Substrate Binding ◽  
Enzymic Activity ◽  
Free Enzyme ◽  
Ligand Concentration ◽  
Double Reciprocal Plot ◽  
Michaelis Constant ◽  
Formal Framework ◽  
Enzyme Species

When information concerning whether or not a ligand interacts with the same enzyme species as do the substrates, the variation of the Michaelis constant Km (for each substrate) with ligand concentration is sometimes used as a diagnostic. It is shown that the Michaelis constant is of no particular value in this respect and may be misleading. Thus, depending on the mechanism, Km may vary with ligand concentration even though the ligand interacts with species far removed in the mechanism from the substrate-binding steps, and it may stay constant in cases where the ligand competes directly for the free enzyme. In contrast, the slope of a double-reciprocal plot of the kinetic data (= Km/Vmax.) (or, equivalently, the ordinate intercept of a Hanes plot A/v versus A, where A is the substrate concentration) independently of the particular mechanism involved uniquely signifies whether or not such interaction occurs. The results clearly indicate that, for purposes other than communicating the substrate concentration yielding control of the enzymic activity, usage of Km and its variation with ligand concentration should be avoided and interest instead focused on the slope, in accordance with the long-established rules of Cleland [Biochim. Biophys. Acta (1963) 67, 188-196], for which the present analysis provides the formal framework.

Production of cephalexin in organic medium at high substrate concentrations with CLEA of penicillin acylase and PGA-450

2007 ◽  
Vol 40 (2) ◽  
pp. 195-203 ◽  
Author(s):  
A. Illanes ◽  
L. Wilson ◽  
C. Altamirano ◽  
Z. Cabrera ◽  
L. Alvarez ◽  
...  
Keyword(s):  
Penicillin Acylase ◽  
Organic Medium ◽  
High Substrate ◽  
Substrate Concentrations

β-GLUCURONIDASE OF RABBIT POLYMORPHONUCLEAR LEUCOCYTES

1950 ◽  
Vol 28e (3) ◽  
pp. 69-79 ◽  
Author(s):  
R. J. Rossiter ◽  
Esther Wong
Keyword(s):  
Enzyme Activity ◽  
Blood Plasma ◽  
Substrate Concentration ◽  
Final Concentration ◽  
White Cell ◽  
Polymorphonuclear Leucocytes ◽  
Michaelis Constant ◽  
Glucuronidase Activity ◽  
Arsanilic Acid ◽  
Optimum Ph

Rabbit polymorphonuclear leucocytes contain an enzyme capable of hydrolyzing biosynthetic phenolphthalein mono-β-glucuronide. The concentration of the enzyme in the white cell is some 2000 times the concentration of the enzyme in the blood plasma. Under the conditions of study, the β-glucuronidase activity was proportional to the concentration of the enzyme. The effect of substrate concentration on the enzyme activity was studied and the Michaelis constant, Ks, determined. The course of the reaction was linear with time for the first 12 hr. and then fell off slightly during the next 12 hr. The optimum pH of the enzyme was 4.45 in either 0.2 M acetate or 0.2 M phthalate buffer. It was not inhibited by cyanide, azide, iodoacetate, fluoride, glycine, thiourea, urethane, arsanilic acid, acetophenone, o-cresol or m-cresol, in a final concentration of 0.01 M. The possible function of β-glucuronidase in rabbit polymorphonuclear leucocytes is discussed.

Initial Studies on the Anaerobic Sequencing Batch Reactor

1992 ◽  
Vol 26 (9-11) ◽  
pp. 2429-2432 ◽  
Author(s):  
R. R. Dague ◽  
C. E. Habben ◽  
S. R. Pidaparti
Keyword(s):  
Substrate Concentration ◽  
Sequencing Batch Reactor ◽  
Batch Reactor ◽  
High Substrate ◽  
Anaerobic Sequencing Batch Reactor ◽  
New Process ◽  
Substrate Conversion ◽  
Solids Separation

This research focuses on an evaluation of the performance of a new process being called the “anaerobic sequencing batch reactor” (ASBR). The ASBR operates on an intermittent, fill and draw regimen. This results in alternating high substrate/low substrate (feast/famine) conditions. The high substrate conditions right after feeding results in high rates of substrate conversion to biogas. The low substrate concentration near the end of the react sequence results in efficient bioflocculation and solids separation.

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