Flow Kinetics of β-Galactosidase Chemically Attached to Nylon Tubing

1975 ◽  
Vol 53 (10) ◽  
pp. 1061-1069 ◽  
Author(s):  
D. Narinesingh ◽  
T. T. Ngo ◽  
K. J. Laidler
Keyword(s):  
Flow Rate ◽  
Substrate Concentration ◽  
Diffusion Control ◽  
Theoretical Treatment ◽  
Enzyme Reactors ◽  
Diffusion Controlled ◽  
Nylon Tubing ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Concentrations

β-Galactosidase (EC 3.2.1.23) has been attached covalently to the inner surface of nylon tubing. An experimental study has been made of the flow kinetics for the hydrolysis of o-nitrophenylgalactose, the substrate concentration and flow rate being varied. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, three different methods of analysis being employed. It is concluded that at the lower substrate concentrations and flow rates employed, the reactions are largely diffusion controlled; with increase in flow rate and substrate concentration the width of the Nernst diffusion layer decreases, and there is found to be less diffusion control. The values of Km(app) vary with flow rate VF, being linear in VF−1/3, and the value extrapolated to very high flow rate agrees well with the Km value for β-galactosidase in free solution. The theory and results are shown to provide guidelines for the design of open tubular heterogeneous enzyme reactors for industrial, biomedical, and analytical applications.

Flow kinetics of immobilized β-glucosidase

1986 ◽  
Vol 64 (2) ◽  
pp. 139-145 ◽  
Author(s):  
Yuchiong Hsuanyu ◽  
Keith J. Laidler
Keyword(s):  
Ph Dependence ◽  
Kinetic Studies ◽  
Free Enzyme ◽  
Diffusion Control ◽  
Michaelis Constant ◽  
Ph Optimum ◽  
Ph Values ◽  
Nylon Tubing ◽  
Kinetics Of ◽  
Substrate Concentrations

The enzyme β-glucosidase was attached covalently to the inner surface of nylon tubing. Flow kinetic studies were carried out at a range of temperatures, pH values, flow rates, and substrate concentrations. Various tests showed that the extent of diffusion control was negligible. At 25 °C the Michaelis constant was 33.4 mM, not greatly different from the value for the enzyme in free solution. The pH dependence was similar to that for the free enzyme. The Arrhenius plots showed inflexions at about 22 °C, as with the free enzyme, the changes in slope being small at the pH optimum of about 5.9 and becoming much more pronounced as the pH is increased or decreased. The immobilized enzyme is more stable than the free enzyme, both on storage at low and higher temperatures, and its reuse stability is greater.

scholarly journals DOES THE KINETICS OF TRYPSIN DIGESTION DEPEND ON THE FORMATION OF A COMPOUND BETWEEN ENZYME AND SUBSTRATE

1922 ◽  
Vol 4 (5) ◽  
pp. 487-509 ◽  
Author(s):  
John H. Northrop
Keyword(s):  
Experimental Evidence ◽  
Substrate Concentration ◽  
Relative Velocity ◽  
Mass Action ◽  
Law Of Mass Action ◽  
Rate Of Hydrolysis ◽  
Gelatin Concentration ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Concentrations

1. The velocity of hydrolysis of gelatin by trypsin increases more slowly than the gelatin concentration and finally becomes nearly independent of the gelatin concentration. The relative velocity of hydrolysis of any two substrate concentrations is independent of the quantity of enzyme used to make the comparison. 2. The rate of hydrolysis is independent of the viscosity of the solution. 3. The percentage retardation of the rate of hydrolysis by inhibiting substances, is independent of the substrate concentration. 4. There is experimental evidence that the enzyme and inhibiting substance are combined to form a widely dissociated compound. 5. If the substrate were also combined with the enzyme, an increase in the substrate concentration should affect the equilibrium between the enzyme and the inhibiting substance. This is not the case. 6. The rate of digestion of a mixture of casein and gelatin is equal to the sum of the rates of hydrolysis of the two substances alone, as it should be if the rate is proportional to the concentration of free enzyme. This contradicts the saturation hypothesis. 7. If the reaction is followed by determining directly the change in the substrate concentration, it is found that this change agrees with the law of mass action; i.e., the rate of digestion is proportional to the substrate concentration.

Kinetics of acetylcholinesterase immobilized on polyethylene tubing

1979 ◽  
Vol 57 (10) ◽  
pp. 1200-1203 ◽  
Author(s):  
T. T. Ngo ◽  
K. J. Laidler ◽  
C. F. Yam
Keyword(s):  
Diffusion Control ◽  
Initial Oxidation ◽  
Diffusion Controlled ◽  
Kinetic Tests ◽  
Acetylthiocholine Iodide ◽  
Carboxylic Groups ◽  
Michaelis Constants ◽  
Polyethylene Tubing ◽  
Kinetics Of ◽  
Hydrolysis Of

Acetylcholinesterase was covalently attached to the inner surface of polyethylene tubing. Initial oxidation generated surface carboxylic groups which, on reaction with thionyl chloride, produced acid chloride groups; these were caused to react with excess ethylenediamine. The amine groups on the surface were linked to glutaraldehyde, and acetylcholinesterase was then attached to the surface. Various kinetic tests showed the catalysis of the hydrolysis of acetylthiocholine iodide to be diffusion controlled. The apparent Michaelis constants were strongly dependent on flow rate and were much larger than the value for the free enzyme. Rate measurements over the temperature range 6–42 °C showed changes in activation energies consistent with diffusion control.

scholarly journals Kinetics of Enzymatic Hydrolysis of Southeast Sulawesi Sago Starch

2020 ◽  
pp. 53-61
Author(s):  
Ansharullah Ansharullah ◽  
Muhammad Natsir
Keyword(s):  
Enzymatic Hydrolysis ◽  
Maximum Velocity ◽  
Enzyme Concentration ◽  
Hydrolysis Rate ◽  
Sago Starch ◽  
Optimum Conditions ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Menten Constant

The aims of this study were to characterize the kinetics of enzymatic hydrolysis of sago starch, obtained from Southeast Sulawesi Indonesia. The enzyme used for hydrolysis was bacterial ∝-amylase (Termamyl 120L from Bacillus licheniformis, E. C. 3.2.1.1).  The method to determine the initial velocity (Vo) of the hydrolysis was developed by differentiation a nonlinear equation (NLE).  The Vo of the hydrolysis was measured at various pH (6.0, 6.5,and 7.0), temperatures (40, 60, 75 and 95oC), enzyme concentrations (0.5, 1.0, 1.5 and 2.0 µg per mL) and in the presence of 70 ppm Ca++. The optimum conditions of this experiment were found to be at pH 6.5 – 7.0 and 75oC, and the Vo increased with increasing enzyme concentration. The Vo values at various substrate concentrations were also determined, which were then used to calculate the enzymes kinetics constant of the hydrolysis, including Michaelis-Menten constant (Km) and maximum velocity (Vmax) using a Hanes plot.  Km and Vmax values were found to be higher in the measurement at pH 7.0 and 75oC. The Km values  at four  different combinations of pH and temperatures (pH 6.5, 40oC; pH 6.5, 75oC; pH 7.0, 40oC; pH 7.0, 75oC) were found to be 0.86, 3.23, 0.77 and 3.83 mg/mL, respectively; and Vmax values were 17.5, 54.3, 20.3 and 57.1 µg/mL/min, respectively. The results obtained showed that hydrolysis rate of this starch was somewhat low.

KINETIC STUDIES ON THE HYDROLYSIS OF BENZOYLCHOLINE BY HUMAN SERUM CHOLINESTERASE

1956 ◽  
Vol 34 (1) ◽  
pp. 637-653 ◽  
Author(s):  
W. Kalow ◽  
K. Genest ◽  
N. Staron
Keyword(s):  
Kinetic Studies ◽  
Reaction Rates ◽  
Serum Cholinesterase ◽  
Initial Reaction ◽  
Ultraviolet Spectrophotometry ◽  
First Order ◽  
Low Concentrations ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Substrate Concentrations

Benzoylcholine stands out from other known substrates of serum cholinesterase because of its high apparent affinity for this enzyme combined with a rapid rate of destruction. The reaction kinetics of the hydrolysis of benzoylcholine can be studied by ultraviolet spectrophotometry, since the absorbance decreases in proportion to the concentration of substrate. Kinetic data obtained by measuring initial reaction rates, and by analyzing continuous hydrolysis curves, are the same within the range of experimental error. The enzymatic data are compatible with the assumption that in the presence of high substrate concentrations a complex consisting of esterase and two substrate molecules is formed. This complex is hydrolyzed more slowly than the complex containing one molecule of substrate which is formed at low concentrations of benzoylcholine. Alkaline hydrolysis of benzoylcholine follows the kinetics of a first order reaction.

scholarly journals Substrate Inhibition in the Hydrolysis of Hippuric Acid Esters by Carboxypeptidase A

1975 ◽  
Vol 53 (2) ◽  
pp. 283-294 ◽  
Author(s):  
Joe Murphy ◽  
John W. Bunting
Keyword(s):  
Substrate Concentration ◽  
Hippuric Acid ◽  
Substrate Inhibition ◽  
Competitive Inhibitor ◽  
Side Chain ◽  
Carboxypeptidase A ◽  
Substrate Activation ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Acid Esters

The dependence of initial velocity upon substrate concentration has been examined in the carboxypeptidase A catalyzed hydrolysis of the following hippuric acid esters (at pH 7.5, 25°, ionic strength O.5): C6H5CONHCH2CO2CHRCO2H: R=CH3; CH2CH3;(CH2)2CH3; (CH2)3CH3; (CH2)5CH3; CH(CH3)2; CH2CH(CH3)2; C6H5; CH2C6H5. All of these esters display marked substrate inhibition of their enzymic hydrolyses. With the exception of R=CH3, the velocity-substrate concentration profiles for each of these esters can be rationalized by the formation of an E.S2 complex which, independent of the alcohol moiety of the ester, reacts approximately 25 times more slowly than the E.S complex. For most of these esters, the formation of E.S2 approximates ordered binding of the substrate molecules at the catalytic and inhibitory sites. While binding at the catalytic site is markedly dependent on the nature of the R group, binding of a second substrate molecule to E.S is not significantly affected by the nature of the R side chain. For R=C6H5, the D ester is neither a substrate nor a competitive inhibitor of the hydrolysis of the L-ester but can replace the L-ester at the binding site which is responsible for substrate inhibition. The kinetic analysis suggests that this behavior of D and L -enantiomers is also typical of the other esters examined (except possibly R=CH3). For R=CH3 only, substrate activation also seems to occur prior to the onset of substrate inhibition at higher substrate concentrations.

Flow kinetics of lactate dehydrogenase chemically attached to nylon tubing

1978 ◽  
Vol 56 (8) ◽  
pp. 774-779 ◽  
Author(s):  
N. J. Daka ◽  
K. J. Laidler
Keyword(s):  
Lactate Dehydrogenase ◽  
Flow Rate ◽  
Rabbit Muscle ◽  
Modified Technique ◽  
Muscle Lactate ◽  
Flow Rates ◽  
Diffusion Controlled ◽  
Michaelis Constants ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Nylon Tubing

Rabbit muscle lactate dehydrogenase (EC 1.1.1.27) was attached covalently to the inner surface of nylon tubing; a modified technique, involving benzidine and glutaraldehyde, was used, and the resulting immobilized enzyme showed no loss of activity over a period of several months. An experimental study was made of the flow kinetics for the reaction between pyruvate and reduced nicotinamide adenine dinucleotide in two limiting cases, one substrate in excess and the concentration of the other one varied. A range of flow rates and temperatures was covered. The results were analyzed in various ways on the basis of the Kobayashi–Laidler treatment of flow systems. It was concluded that the kinetics are largely diffusion-controlled, especially at the lower substrate concentrations and flow rates. The values of the apparent Michaelis constants vary with flow rate vf, being linear in vf−1/3, and the values extrapolated to infinite flow rate (vf−1/3 = 0) approach the values for the enzyme in free solution. Analysis of the rates led to activation energies for the diffusion of the two substrates.

Rate and equilibrium constants for formation and hydrolysis of 9-formylfluorene oxime: diffusion-controlled trapping of a protonated aldehyde by hydroxylamine

2000 ◽  
Vol 78 (12) ◽  
pp. 1594-1612 ◽  
Author(s):  
RA More O'Ferrall ◽  
D M O'Brien ◽  
D G Murphy
Keyword(s):  
Aqueous Solution ◽  
Acetic Acid ◽  
Reaction Pathway ◽  
Equilibrium Constants ◽  
Diffusion Control ◽  
General Measure ◽  
Hydroxide Ion ◽  
Diffusion Controlled ◽  
Enol Tautomer ◽  
Hydrolysis Of

Equilibrium constants Kadd = 440 and Kox = 3.0 × 108 for formation of a carbinolamine adduct and oxime, respectively from 9-formylfluorene and hydroxylamine, and pKa = –1.62 for protonation of the oxime, have been evaluated at 25°C in aqueous solution, based on measurements in hydroxylamine buffers, acetic acid buffers, and dilute HCl. Rate constants for hydrolysis of the oxime have been measured in the acidity range pH 4–12 M HClO4. At the highest acidities, a reaction pathway via protonated carbinolamine has been identified: evidence is presented that the reverse of this reaction involves rate-determining attack of hydroxylamine upon protonated 9-formylfluorene. By assuming that the attack of hydroxylamine is diffusion-controlled, with rate constant 3 × 109 M –1 s–1, a pKa for O-protonation of the aldehyde (–4.5) is derived. Taking account of the equilibrium constant for enolization of 9-formylfluorene (KE = 16.6), a pKa for for C-protonation of the enol tautomer ((–5.7) may also be obtained. Comparison of this pKa with that of the enol of acetophenone shows that the enol of 9-formylfluorene is less basic by a factor of 1010. By combining pKas for protonation of the aldehyde and oxime with measured or estimated equilibrium constants for addition of water, hydroxide ion, and hydroxylamine to 9-formylfluorene, it is also possible to obtain values of pKR = –5.3, 4.1, and 12.25 for the protonated 9-formylfluorene, protonated oxime, and 9-formylfluorene, respectively. The usefulness of pKR in providing a general measure of equilibrium constants for electrophile-nucleophile combination reactions is discussed.Key words: oxime, formyfluorene, hydrolysis, protonation, diffusion-control.

scholarly journals THE KINETICS OF TRYPSIN DIGESTION

1924 ◽  
Vol 6 (4) ◽  
pp. 429-437 ◽  
Author(s):  
John H. Northrop
Keyword(s):  
High Temperature ◽  
Substrate Concentration ◽  
Trypsin Digestion ◽  
Velocity Constant ◽  
Rate Of Hydrolysis ◽  
Kinetics Of ◽  
Hydrolysis Of

1. A study has been made of the rate of hydrolysis of concentrated gelatin solutions at a high temperature and with a large amount of trypsin. 2. Under these conditions the substrate concentration may be considered constant and the only variable is the decrease in the amount of trypsin owing to inactivation. 3. The theory based on the mass law predicts that under these conditions (a) the rate at any time will be proportional to the concentration of trypsin at that time; (b) the reaction should approximate a monomolecular one if the total hydrolysis observed is taken as the amount of substrate available; (c) that the velocity constant calculated in this way should agree with the constant for the decomposition of the enzyme and that it should be independent of the concentration of enzyme instead of proportional to it as is usually the case; and (d) that the total amount of substrate decomposed should be proportional to the amount of trypsin added at the beginning instead of independent of it. These results have been obtained experimentally.

Sign in / Sign up

Export Citation Format

Share Document