BTX Degradation: The concept of microbial integration

2019 ◽  
Author(s):  
Chem Int
Keyword(s):  
Gas Chromatography ◽  
Soil Sample ◽  
High Activity ◽  
Niger Delta ◽  
Batch Reactor ◽  
Free Enzyme ◽  
Delta Area ◽  
Substrate Complex ◽  
Fungal Activity ◽  
Enzyme Substrate

The concept of microbial integration was carried out to examine bacterial and fungal activity on bezene, toluence and xylene (BTX) degradation in a batch reactor. The investigation was conducted for thirty five day of exposure of contact of members and substrate which yielded enzyme substrate complex as well disintegrated to produce products and free enzyme. Bacterial and fungal concentration was monitored per week and the results obtained recorded. The gas chromatography results of Ngara soil sample investigated reveals the concentration of M, P, and O – Xylene for different days of exposure. Increase in both bacterial and fungal was experienced with decrease in BTX concentration, whereas increase in bacterial is more than fungi, indicating the high activity of bacterial in the reactor than that of fungi. Although, both were well integrated in bioremediation program to enhance the effective remediation of BTX contaminants in Ngara soil, Omuigwe Alun Community, Niger Delta Area of Nigeria.

The influence of pH and inhibitors on the kinetics of enzyme reactions involving two intermediates

1967 ◽  
Vol 45 (5) ◽  
pp. 539-546 ◽  
Author(s):  
Harvey Kaplan ◽  
Keith J. Laidler
Keyword(s):  
Steady State ◽  
Ph Dependence ◽  
Free Enzyme ◽  
State Equations ◽  
Enzyme Reactions ◽  
Substrate Complex ◽  
Rate Determining Step ◽  
Enzyme Substrate ◽  
Special Cases ◽  
Influence Of Ph

General steady-state equations are worked out for enzyme reactions which occur according to the scheme [Formula: see text]Equations showing the pH dependence of the kinetic parameters are developed in a form which distinguishes between essential and nonessential ionizing groups. The pK dependence of [Formula: see text], the second-order constant extrapolated to zero substrate constant, gives pK values for groups which ionize on the free enzyme, but reveals such a pK only if the corresponding group is also involved in the breakdown of the Michaelis complex. General steady-state equations are also developed for the case in which an inhibitor can combine with the free enzyme, the enzyme–substrate complex, and also a second intermediate (e.g. an acyl enzyme). The equations are given in a form that is convenient for analyzing the experimental results, and a number of special cases are considered. It is shown how the type of inhibition depends not only on the nature of the inhibitor but also on that of the substrate, an important factor being the rate-determining step of the reaction. Examples of the various kinds of behavior are given.

Total Enzyme-substrate Complex Which Includes Product-destined Enzyme-substrate Complex

2019 ◽  
pp. 1-7
Author(s):  
Ikechukwu I. Udema
Keyword(s):  
Complex Formation ◽  
Maximum Velocity ◽  
Free Enzyme ◽  
Theoretical Research ◽  
Substrate Complex ◽  
Continuous Formation ◽  
Enzyme Substrate ◽  
The Difference ◽  
Total Enzyme

Background: There is no much interest in the determination of total enzyme-substrate complex concentration ([ES]T) which includes undissociated ES that is unaccounted for unlike the usual ES destined for transformation into free enzyme and product or substrate. The reason is speculatively as a result of the lack of awareness of such possibility via sequestration. Objectives: 1) To derive on the basis of both reverse – and standard – quasi-steady – state assumptions equations for the determination of [ES]T which is not restricted to the complex which dissociates to product/substrate and free enzyme and 2) quantitate the value of [ES]T. Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to calculate relevant parameters. Results: The [EST] is < [E] ( i. e. [ET] - [ES]); [EST] decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while the velocity of amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST]. Conclusion: The equations for the determination of the total enzyme-substrate complex, free enzyme without any complex formation before and after dissociation of enzyme-complex into product and/or substrate and free enzyme were derived. The difference, [ET] - [ES] is a heterogeneous mixture of undissociated ES and free enzyme without any complex formation. This is the case because [ES] which dissociates into product is only a part of the total enzyme-substrate complex. There is a continuous formation of ES during and at the expiry of the duration of assay as long as there is no total substrate depletion.

Final Phase of Enzyme Reactions Following a Michaelis-Menten Mechanism in which the Free Enzyme and/or the Enzyme-Substrate Complex are Unstable

1994 ◽  
Vol 375 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Ramón Varón ◽  
Carmelo Garrido del Solo ◽  
Manuela Garcίa-Moreno ◽  
Angela Sánchez-Gracia ◽  
Francisco Garcίa-Cánovas
Keyword(s):  
Free Enzyme ◽  
Final Phase ◽  
Enzyme Reactions ◽  
Substrate Complex ◽  
Enzyme Substrate

scholarly journals Kinetic analysis of a Michaelis-Menten mechanism in which the enzyme is unstable

1993 ◽  
Vol 294 (2) ◽  
pp. 459-464 ◽  
Author(s):  
C Garrido-del Solo ◽  
F García-Cánovas ◽  
B H Havsteen ◽  
R Varón-Castellanos
Keyword(s):  
Data Analysis ◽  
Experimental Design ◽  
Numerical Simulations ◽  
Kinetic Analysis ◽  
Kinetic Data ◽  
Time Course ◽  
Enzyme Concentration ◽  
Free Enzyme ◽  
Substrate Complex ◽  
Enzyme Substrate

A kinetic analysis of the Michaelis-Menten mechanism is made for the cases in which the free enzyme, or the enzyme-substrate complex, or both, are unstable, either spontaneously or as a result of the addition of a reagent. The explicit time-course equations of all of the species involved has been derived under conditions of limiting enzyme concentration. The validity of these equations has been checked by using numerical simulations. An experimental design and a kinetic data analysis allowing the evaluation of the parameters and kinetic constants are recommended.

scholarly journals The Experimentally Determined Velocity of Catalysis could be Higher in the Absence of Sequestration

2019 ◽  
pp. 1-12
Author(s):  
Ikechukwu I. Udema ◽  
Abraham Olalere Onigbinde
Keyword(s):  
Maximum Velocity ◽  
Order Rate Constant ◽  
Free Enzyme ◽  
Theoretical Research ◽  
Coefficient Of Determination ◽  
First Order ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Pseudo First Order ◽  
The Relationship

Background: It is not unusual to observe calculated “total” free enzyme ([E]) in enzyme catalysed reaction, but this should include total enzyme-substrate complex ([EST]) which accounts for sequestration. Objectives: 1) To show indirectly that the velocities of catalytic action can be higher than experimentally observed velocities without sequestration and 2) redefine the relationship between velocity of hydrolysis with Michaelian enzyme and [E], where concentration of substrate, [ST] <  Michaelis-Menten constant, KM. Methods: A theoretical research and experimentation using Bernfeld method to determine velocities of amylolysis with which to mathematically calculate [EST] and the enzyme-substrate complex ([ES]) prepared for product, P, formation. Results: The [EST] is < [E]; [EST] and pseudo-first order constant, k decreased with increasing [ST] and increased with increasing concentration of enzyme [ET] while velocity amylolysis, v and maximum velocity of amylolysis, vmax expectedly increased with increasing [ET] and [ST]. Conclusion: The fact is that the [EST] is lower than what is usually referred to as free enzyme ([ET] - [ES]). Therefore, if the additional part of [EST] dissociated into product within the duration of assay, the velocity of amylolysis could be higher. The most important outcome and corollary when [KM] > [ST] is that v a 1/[E], v a [E][ST] and a quadratic relationship exists between pseudo-first order rate constant and maximum velocity of amylolysis; separately, v is not a [E] and if v a [ST] (if v/[ST] is constant with coefficient of determination = 1), then KM is not applicable.

pH-dependence of the fast step of maltose hydrolysis catalysed by glucoamylase G1 from Aspergillus niger

2000 ◽  
Vol 349 (2) ◽  
pp. 623-628 ◽  
Author(s):  
Ulla CHRISTENSEN
Keyword(s):  
Aspergillus Niger ◽  
Ph Dependence ◽  
Acid Group ◽  
Reaction Scheme ◽  
Free Enzyme ◽  
Pka Values ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Range ◽  
State Kinetic

The presteady-state kinetic parameters of the interaction of wild-type glucoamylase from Aspergillus niger (EC 3.2.1.3) with maltose were obtained and analysed in the pH range 3-7 with intervals of 0.25 pH units. In all cases the following three-step reaction scheme was found to apply. E+S ES1 ES2 E+P The general result of the analysis of the presteady-state kinetics is that glucoamylase G1 is affected by the protonation states of three groups, with pKa values of 2.7, 4.5 and 5.7 in the free enzyme and of 2.7, 4.75 and 6.5 in the first enzyme-substrate complex. The protonation of the group in the enzyme-substrate complex with a pKa 6.5 had no effect on k2 (1640 s-1) or k-2 (20±4 s-1), but resulted in a stronger enzyme-substrate interaction, due to a decrease of K1 from 40 to 6.3 mM. In other words, when the substrate is bound, the pKa of the acid group changes to increase the fraction of reactive enzyme. Since this pKa parallels that of the Michaelis complex, known from the pH-dependence of kcat, the group in question is most probably the catalytic acid Glu-179. Protonation of Glu-179 thus is of no importance in the second step, clearly indicating that this step represents a conformational change and not the actual hydrolysis step of the reaction. Protonation of the pKa = 4.75 group leads to a small decrease in k2 to 1090 s-1, and also to minor changes in K1. The group with pKa = 2.7 leads to a major decrease of k2, of which the limit may be zero, but shows no effect on K1. Thus no difference is seen between the pKa values of the free enzyme and of the first enzyme-substrate complex at low pH.

scholarly journals Substrate- and alkali-metal-ion-induced pK shifts in intestinal brush-border sucrase, according to the three-protons model

1989 ◽  
Vol 258 (1) ◽  
pp. 41-48 ◽  
Author(s):  
M Vasseur ◽  
G van Melle ◽  
F Alvarado
Keyword(s):  
Metal Ion ◽  
Enzyme Activation ◽  
Free Enzyme ◽  
Molecular Constant ◽  
Double Logarithmic Plot ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Unequal Number ◽  
Concentration Factors ◽  
Kinetic Effects

To define adequately enzyme activation/inhibition mechanisms as a function of pH, it is necessary to characterize the effector-induced pK shifts on both the free enzyme and on the enzyme-substrate complex. On the basis of our recent three-protons model for sucrase [Vasseur, van Melle, Frangne & Alvarado (1988) Biochem. J. 251, 667-675], we show how the ‘fundamental’ pK values, deduced from the classical double-logarithmic transformations, are insufficient to generate the required information. This insufficiency derives from the fact that, for sucrase, the acid ionization constant, K1, is a molecular constant that involves complex, V-type plus K-type, activatory and inhibitory kinetic effects. As a consequence, substrate-induced pK shifts cannot be interpreted correctly only by using the fundamental pK approach, because an unequal number of key protons is involved, depending on whether the free enzyme or the enzyme-substrate complex is considered. We demonstrate how this problem can be solved by using the ‘theoretical’ pK values, derived from the reciprocals of the Michaelis pH functions, i.e. Cha's fractional concentration factors. The procedure we propose, which is general, has the advantage of yielding all the macroscopic pK values for any given model, as calculated from the microscopic pK values. Furthermore, it permits predicting pK shifts as a function of [S] and/or [A] (where S is the substrate and A is the allosteric modifier), an objective that cannot be attained by using the double-logarithmic plot approach. Finally, we describe the relation existing between the fundamental and the theoretical pK values.

scholarly journals Kinetic Analysis of Guanidine Hydrochloride Inactivation of β-Galactosidase in the Presence of Galactose

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Charles O. Nwamba ◽  
Ferdinand C. Chilaka
Keyword(s):  
Rate Constants ◽  
Initial Velocity ◽  
Competitive Inhibition ◽  
Guanidine Hydrochloride ◽  
Free Enzyme ◽  
Velocity Data ◽  
Substrate Complex ◽  
Inactivation Rate Constant ◽  
Enzyme Substrate ◽  
Straight Lines

Inactivation of purified β-Galactosidase was done with GdnHCl in the absence and presence of varying [galactose] at 50°C and at pH 4.5. Lineweaver-Burk plots of initial velocity data, in the presence and absence of guanidine hydrochloride (GdnHCl) and galactose, were used to determine the relevant Km and Vmax values, with p-nitrophenyl β-D-galactopyranoside (pNPG) as substrate, S. Plots of ln([P]∞−[P]t) against time in the presence of GdnHCl yielded the inactivation rate constant, A. Plots of A versus [S] at different galactose concentrations were straight lines that became increasingly less steep as the [galactose] increased, showing that A was dependent on [S]. Slopes and intercepts of the 1/[P]∞ versus 1/[S] yielded k+0 and k'+0, the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of k+0 and k'+0 versus [galactose] showed that galactose protected the free enzyme as well as the enzyme-substrate complex (only at the lowest and highest [galactose]) against GdnHCl inactivation. In the absence of galactose, GdnHCl exhibited some degree of non-competitive inhibition. In the presence of GdnHCl, galactose exhibited competitive inhibition at the lower [galactose] of 5 mM which changed to non-competitive as the [galactose] increased. The implications of our findings are further discussed.

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

scholarly journals Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

2021 ◽  
Vol 14 ◽  
pp. 117863612110246
Author(s):  
Cheuk Yin Lai ◽  
Ka Lun Ng ◽  
Hao Wang ◽  
Chui Chi Lam ◽  
Wan Keung Raymond Wong
Keyword(s):  
Escherichia Coli ◽  
Cellulolytic Bacterium ◽  
Systematic Screening ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycosylation Sites ◽  
Endogenous Proteins ◽  
Cellulose Binding

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

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