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Enzyme kinetics: Effect of immobilization on kinetic parameters

Enzyme kinetics: Effect of immobilization on kinetic parameters


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What is the typical effect of enzyme immobilization on the kinetic parameters of an enzyme's activity?

Can one assume that they'd stay approximately the same or is there a gross change? Any way to estimate the effect?

The native parameters are as follows:

kcat 0.5 1/min Km 0.6 microM [E0] 5 micro M [S] 60 micro M

Could I expect to retain them after immobilization? Or are these too high for an immobilized enzyme?

If it matters, it is a 500 residue enzyme with a MW of approx. 65,000 Da


The main factor influencing the kinetics of immobilized enzymes is thought to be the rate of diffusion of substrate and product towards and away from the enzyme, respectively.

This has been discussed on ResearchGate and an article in Process Biochemistry from one of those in the discussion quotes figures for effects on immobilized laccase: approx. 100x increase in Km and 2-5x reduction in Vmax (as noted in the poster's comment).

There are several theoretical treatments of the question:


Effect of immobilization on the activity of catalase carried by poly(HEMA-GMA) cryogels

Hydrogen peroxide is converted by catalase to molecular oxygen and water to remove oxidative stress. In this study, catalase immobilization was performed using poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) (poly(HEMA-GMA)) cryogels with different amounts of GMA. Catalase adsorption capacity of 298.7 ± 9.9 mg/g was achieved at the end of 9 h using the poly(HEMA-GMA)-250 cryogel. Kinetic parameters and the inhibitory effects of pesticides such as 4,4'-DDE and 4,4'-DDT on the activity of free and immobilized catalase enzyme were investigated. While the Vmax value of the immobilized enzyme was reduced 4-fold compared to the free enzyme, in the case of the comparison of the KM values, the affinity of the immobilized enzyme was increased by 1.94 times against the substrate. The inhibitory effect of 4,4'-DDT pesticide was found to be higher for the immobilized and free enzyme. NaCl (1 M, pH: 7.0) solution was used for desorption of the adsorbed catalase enzyme. A desorption ratio of 96.45% was achieved. The technique used in this study is promising regarding for the immobilization of catalase enzyme to increase the operational activity. Therefore, poly(HEMA-GMA) cryogels have the potential to be used for immobilization of catalase enzyme in the fields of biology and biochemistry.

Keywords: Catalase Immobilization Operational activity.


Microenvironmental effects on enzyme catalysis. Kinetic study of polyanionic and polycationic derivatives of chymotrypsin

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Microenvironmental effects on enzyme catalysis. Kinetic study of polyanionic and polycationic derivatives of chymotrypsin

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

Note: In lieu of an abstract, this is the article's first page.


Abstract

Porous silicon matrixes are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. In an effort to gain a quantitative understanding of the effects of immobilization on enzyme activity, we compared the activity of glutathione-S-transferase immobilized in electrochemically etched porous silicon films (∼6.5 μm thick) with the enzyme in solution. Kinetic measurements were made by varying the glutathione concentration while maintaining a fixed saturating concentration of 1-chloro-2,4-dinitrobenzene. The reaction kinetics follow steady-state equilibrium behavior. The specific activity of the free enzyme in solution is ∼4× higher than the immobilized enzyme, for which we measured an apparent Km GSH value of 1.0 ± 0.3. The maximum velocity, Vmax, is linearly proportional to immobilized enzyme concentration, but the magnitude is ∼20 times lower than that in solution. Results suggest ∼25% of the enzyme is bound with the catalytic site in an inactive conformation or in a hindered orientation. Finally, the effects of hydration and exposure to denaturants on the immobilized enzyme activity are presented.


Description

Applied Biochemistry and Bioengineering, Volume 1: Immobilized Enzyme Principles focuses on the utilization of immobilization techniques for the study and application of enzyme catalysts in a variety of potential end-uses. This book emphasizes the preparation of enzyme-support systems, effects caused by the concurrent phenomena of enzyme-catalyzed reaction kinetics and mass transfer resistances, and how these reactions are incorporated into the design of enzyme-catalyzed reactor systems. The magnitude of the perturbation of the apparent kinetic parameters of an immobilized enzyme that could serve in principle as a measure of the effective concentrations of substrate, modifier, or inhibitor at the site of the enzymic reaction is also explained. This volume is recommended for biological scientists and engineers, as well as researchers interested in the biochemical common denominator that causes the interaction of engineering practice and biological sciences for technological development.


Abstract

The use of liquid enzymes for the production of biodiesel as an alternative to chemical catalysts requires significant investigation due to the lack of experimental data for the various feedstock and catalyst combinations. In this paper, reaction rates and kinetic modeling of the transesterification of castor oil with methanol using the enzyme Eversa Transform as the catalyst were investigated. Reactions were carried out for 8 h at 35 °C with an alcohol-to-oil molar ratio equal to 6:1, a 5 wt % of liquid enzyme solution, and addition of 5 wt % of water by weight of castor oil. From the concentration data, four different reaction mechanistic models were compared to determine the mechanism that best fitted the experimental data. Mechanisms where the methanolysis and hydrolysis reactions occurred simultaneously in the system were best at describing the concentration profiles. The high methanolysis rates of glycerides that were obtained indicated that transesterification dominates over hydrolysis. The mechanism among the four models proposed that gave the best fit could be simplified, eliminating the kinetic parameters with negligible effects on the reaction rates. This model was able to fit the experimental data at different reaction temperatures.


Michaelis &ndash Menten Equation

Leonor Michaelis and Maud Menten postulated that the enzyme first combines reversibly with its substrate to form an enzyme-substrate complex in a relatively fast reversible step:


Eqn.1

In the next step, this ES complex is breaks down in to the free enzyme and the reaction product P:

Eqn.2

Since the second step is the rate limiting step, the rate of overall reaction must be proportional to the concentration of the ES that reacts in the second step. The relationship between substrate concentration, [S] and Initial velocity of enzyme, V0 (Fig. 1) has the same general shape for most enzymes (it approaches a rectangular hyperbola). This can be expressed algebraically by the Michaelis-Menten equation. Based on their basic hypothesis that the rate limiting step in enzymatic reactions is the breakdown of the ES complex to free enzyme and product, Michaelis and Menten derived an equation which is

Eqn.3

The necessary terms in this reaction are [S], V0, Vmax, and Km (Michaelis constant),. All these terms can be measured experimentally.

Lineweaver &ndash Burke plot


In 1934, Lineweaver and Burke made a simple mathematical alteration in the process by plotting a double inverse of substrate concentration and reaction rate.


Eqn.4


For enzymes obeying the Michaelis-Menten relationship, the &ldquodouble reciprocal&rdquo of the V0 versus [S] from the first graph,(fig1) yields a straight line (Fig. 2). The slope of this straight line is KM /Vmax, which has an intercept of 1/Vmax on the 1/V0 axis, and an intercept of -1/KM on the 1/[S] axis. The double-reciprocal presentation, also called a Lineweaver-Burk plot. The main advantage of Lineweaver-Burk plot is to determine the Vmax more accurately, which can only be approximated from a simple graph of V0 versus [S] (Fig 1).



Fig2: Lineweaver-Burk plot.

Adapted from David L. Nelson, Michael M. Cox , Lehninger principles of biochemistry, 4th edition.


What is the true enzyme kinetics in the biological system? An investigation of macromolecular crowding effect upon enzyme kinetics of glucose-6-phosphate dehydrogenase

Enzyme kinetic parameters for rate equations are vital in metabolic network simulation, a major part of systems biology research efforts. Measurements of Michaelis-Menten kinetic parameters Km and Kcat have been performed for enzymes glucose-6-phosphate dehydrogenase (G6P DH) under crowded conditions using molecular crowding agents bovine serum albumin (BSA) and polyethylene glycol (PEG) of 8000 Da molecular weight. An increase in Kcat was observed at very low concentrations of crowding agent, and also at high crowder concentrations when the experiment was performed at 45 °C with PEG. The observed pattern in Kcat for G6P DH at high crowder concentrations has been explained via modelling using excluded volume theory. An increase in rate was observed at 45 °C for G6P DH versus 30 °C this has been modelled via the Arrhenius equation.


Kinetic Characterization and Effect of Immobilized Thermostable β-Glucosidase in Alginate Gel Beads on Sugarcane Juice

A thermostable β-glucosidase was effectively immobilized on alginate by the method of gel entrapment. After optimization of immobilized conditions, recovered enzyme activity was 60%. Optimum pH, temperature, kinetic parameters, thermal and pH stability, reusability, and storage stability were investigated. The

for immobilized β-glucosidase were estimated to be 5.0 mM and 0.64 U/ml, respectively. When comparing, free and immobilized enzyme, change was observed in optimum pH and temperature from 5.0 to 6.0 and 60°C to 80°C, respectively. Immobilized enzyme showed an increase in pH stability over the studied pH range (3.0–10.0) and stability at temperature up to 80°C. The storage stability and reusability of the immobilized β-glucosidase were improved significantly, with 12.09% activity retention at 30°C after being stored for 25 d and 17.85% residual activity after being repeatedly used for 4 times. The effect of both free and immobilized β-glucosidase enzyme on physicochemical properties of sugarcane juice was also analyzed.

1. Introduction

β-Glucosidase (β-D-glucoside glucohydrolase EC 3.2.1.21) is a part of multienzyme cellulase complex, whose synthesis and action are intricately controlled by regulatory mechanisms in the organisms that produce these enzymes. The enzymatic hydrolysis of cellulose involves three types of cellulase activities (cellobiohydrolases, endoglucanases, and β-glucosidases) working in synergy [1, 2].

β-Glucosidases hydrolyze β-D-glycosidic bond to release nonreducing β-D-glucose residue and terminal aglycone. These are widely used in the various biotechnological processes including aroma and flavour enrichment [3], discoloration of fruit juices prevention [4], and organoleptic properties of citrus fruit juices improvement, in which the bitterness is in part due to a glucosidic compound, naringin (4,5,7-trihydroxyflavanone-7-rhamnoglucoside) [5]. β-Glucosidase also acts as a key enzyme in the enzymatic release of aromatic compounds from glucosidic precursors present in fruits and fermentation products [6]. Transglycosylation reactions by β-glucosidase have great importance in wine or beverage industry because of their abilities to improve the aroma [7]. The synthetic activity of β-glucosidase can be used in the preparation of a variety of compounds such as oligosaccharides and glycoconjugates that have potential for use as agrochemicals and drugs. β-Glucosidase, produced intracellularly by many microorganisms, usually shows a broad specificity and also transferase activity [8].

Nevertheless, the applications of enzyme in industry remain limited due to the high production cost, stability, and need for repeated enzyme purification. The main strategy to increase the enzyme stability and reusability is the immobilization of enzyme. Some of the more significant advantages of immobilized enzymes over their soluble counterparts include the enhanced stability under extreme conditions of temperature, pH, and organic solvents recovery and subsequent applicability to continuous processes [9].

Alginate, a polysaccharide consisting of glucuronic acid and mannuronic acid moieties, has been found to be a matrix of priority due to its biocompatibility and processivity [10]. It is a reversibly soluble insoluble polymer which changes solubility in the presence of calcium [11]. To date, cross-linked alginate has been successfully used for encapsulation of many biological molecules [12, 13].

The present study describes the immobilization of β-glucosidase in alginate gel beads and the effect of this immobilization on kinetic characteristics of immobilized β-glucosidase in comparison with free enzyme. For application purposes, the effect of both free and immobilized β-glucosidase enzymes on physicochemical properties of sugarcane juice was also analyzed.

2. Material and Methods

2.1. Chemicals and Bacterial Culture

All chemicals, media, and components used were of analytical grade and obtained from Sigma Chemicals Ltd., Himedia Laboratories Ltd., GeNei, SRL, and Merck Pvt. Ltd. Recombinant β-glucosidase from Bacillus subtilis strain PS (identified using 16S rDNA sequencing GenBank Accession number JQ066263) cloned in E. coli DH5α in our laboratory was used in this study [14]. The cloned β-glucosidase produces extracellularly by the bacterial cell.

2.2. Production and Partial Purification of β-Glucosidase

E. coli DH5α containing recombinant enzyme was cultured in Luria broth in 250 mL conical flasks using incubator shaker (150 rpm) at 37°C for 12 h. For extraction of extracellular β-glucosidase enzyme, cells were harvested at 10,000 rpm for 30 minutes at 4°C and supernatant was collected and assayed for β-glucosidase activity. β-Glucosidase enzyme was purified partially using ammonium sulfate fractionation followed by dialysis. All the purification steps were performed at 4°C. Crude enzyme in the cell free supernatant was precipitated by adding ammonium sulfate up to 70%. The precipitates were separated by centrifugation and resuspended in acetate buffer (100 mM, pH 5.0) and dialyzed against the same buffer overnight with two buffer changes. The β-glucosidase activity after dialysis was measured and it was used for further studies.

2.3. Immobilization of β-Glucosidase in Ca-Alginate Gel Beads

Calcium alginate gel beads were prepared as described by Busto et al. [15]. Briefly, sodium alginate solution, 1%, 2%, 3%, 4%, and 5%, was prepared in a suitable amount of enzyme. This solution was dropped in 0.05 M, 0.1 M, and 0.2 M calcium chloride solution under continuous stirring. The beads were cured for 1–5 hrs in the calcium chloride solution, washed several times with a 0.03 M CaCl2 solution until no enzyme activity was observed in the final washing, and stored at 4°C in this solution.

2.4. Determination of β-Glucosidase Enzyme Activity

β-Glucosidase activity was evaluated spectrophotometrically using pNPG as an artificial substrate. The reaction mixture, containing 100 μL β-glucosidase enzyme extract in acetate buffer (pH 5.0, 100 mM) and 100 μL of pNPG in similar buffer, was incubated for 30 min at 60°C. The reaction was stopped by adding 2 mL of 1 M Na2CO3 solution and the absorbance was measured at λ405 nm [16]. The activity of immobilized β-glucosidase was determined using the procedure given above except 0.2 g immobilized enzyme was used in place of 100 μL enzyme extract.

2.5. Kinetic Characterization of Immobilized and Free Enzyme

The kinetic constants of Michaelis values ( and

) for the free and immobilized enzyme preparations were determined using Lineweaver-Burk plot by measuring the enzymatic activity at different substrate concentrations (1–15 mM). The turnover number and catalytic efficiency were also determined.

2.6. Effect of pH and Temperature on Free and Immobilized β-Glucosidase Activity

The optimum pH for β-glucosidase activity was studied over a pH range of 3 to 10 to determine the activity of free as well as immobilized enzyme. Citrate buffer (pH 3–6), phosphate buffer (pH 7-8), and glycine-NaOH buffer (pH 9-10) were used to determine enzyme activity. The optimum temperature for β-glucosidase activity was determined by incubating the reaction mixture over the temperature range of 40–80°C at the optimum pH.

2.7. Effect of pH and Temperature on Stability of Extracellular Free β-Glucosidase

The stability of the enzyme was determined by preincubating the enzyme for 30 min at 37°C with various buffers having a pH range of 3 to 10 as mentioned earlier. After incubation, the residual enzyme activity (%) was measured using acetate buffer (pH 5.0) as explained earlier. The thermal stability of the enzyme was studied by preincubating the enzyme at different temperatures ranging from 40 to 80°C for 0–120 min at optimum pH.

2.8. Storage Stability and Reusability of Immobilized β-Glucosidase

The storage stability of immobilized β-glucosidase at 4°C and 30°C was measured by calculating the residual activity at the interval of 5 d up to 30 d. Reusability of immobilized enzyme was also investigated by measuring its activity after repeated cycles of use.

2.9. Estimation of Reducing Sugar in Sugarcane Juice

Sugarcane juice was incubated at 60°C for 30 min in the presence of free and immobilized β-glucosidase in alginate gel and the reducing sugar was estimated by Somogyi’s method [17]. Determination of relative density and viscosity coefficient of treated sugarcane juice with free and immobilized enzyme was done by density bottle method and Ostwald’s viscometer.

2.10. Absorption Spectra of Sugarcane Juice

Sugarcane juice was incubated for 30 min at 60°C in the presence of free and immobilized β-glucosidase and then the samples were diluted to five times with distilled water. The absorption spectra of untreated and treated juice were analyzed in visible range (400–700 nm).

2.11. Statistical Analysis

The mean values and standard deviation of three experiments were calculated and presented on the figures as error bars. One-way ANOVA at the significance levels of 0.005 and 0.001 was performed using Microsoft excel 2007 statistical tools.

3. Results and Discussion

3.1. Immobilization of β-Glucosidase in Gel

The effects of Na-alginate and CaCl2 concentrations on the bead formation and immobilization of enzyme revealed that maximum immobilization efficiency (60% β-glucosidase activity) was obtained with 3% sodium alginate and 0.2 M CaCl2 for 1 h (Figure 1). A similar high level of activity was obtained by Ortega et al. [18], where β-glucosidase from Aspergillus niger was immobilized in calcium alginate, using an alginate concentration of 3%. No significant effect was observed on the β-glucosidase immobilization with various concentrations of the immobilization time. On increasing CaCl2 concentration from 0.05 to 0.2 M, the stability of the gel increased without loss of enzyme activity, which was in agreement with the results obtained by Jain and Ghose [19]. Lower immobilization efficiency of β-glucosidase at sodium alginate concentrations below 3% was suggested to be due to larger pore sizes of the less tightly cross-linked gels [20], and at sodium alginate concentrations above 3% might be due to lack of uniform pore size because of high viscosity of the enzyme alginate mixture.


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(e)
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(e) Immobilization of β-glucosidase in different concentrations of sodium alginate ((a) 1% sodium alginate (b) 2% sodium alginate (c) 3% sodium alginate (d) 4% sodium alginate (e) 5% sodium alginate).
3.2. Kinetic Characteristics of Free and Immobilized β-Glucosidase

The effect of the substrate concentration on the rate catalyzed by free and immobilized enzyme was studied using varying concentrations (1–15 mM) of pNPG as the substrate. Michaelis constant ( ) and the maximum reaction velocity ( ) of free and immobilized enzyme were calculated from the Lineweaver-Burk plot. The immobilized enzyme in alginate showed an apparent value (

mM) higher than the free enzyme (

mM). Similar results were reported by Quiroga et al. [21] for immobilized araujiain, a cysteine phytoprotease in calcium alginate gel beads. An increase in after immobilization indicates that the immobilized enzymes have an apparent lower affinity for their substrate than the free enzyme. This may be caused by the support steric hindrance of the active site, by the loss of enzyme flexibility necessary for substrate binding, or by diffusional resistance to substrate transport [22]. In addition, a decrease in was observed for immobilized enzyme (0.745 μmol min −1 mL −1 ) as compared to free enzyme (0.94 μmol min −1 mL −1 ). This decrease might be attributed to limited accessibility of substrate molecules to the active sites of the enzyme and the interaction of the enzymes with the functional groups on the surface of beads or large areas of contact between enzyme and support. The apparent

values of free and immobilized β-glucosidase under standard assay conditions were

s −1 and s −1 , respectively. The catalytic efficiency of free and immobilized β-glucosidase was

3.3. Effect of Temperature on Free and Immobilized β-Glucosidase

Effect of temperature variations on free and immobilized enzyme activity was investigated. Reactions were carried out at pH 6.0 and temperature influence was studied within the 40–90°C range (Figure 2). The optimum temperature of the free enzyme was 60°C but after the entrapment process a shift in such temperature was observed and the immobilized enzyme exhibited the highest activity at 80°C in gel, since hydrophobic and other secondary interactions of the immobilized enzyme might impair conformational flexibility needing higher temperatures for the enzyme molecule to recognize and attain a proper conformation in order to keep its reactivity [23]. Thereafter, a loss in activity above 80°C might be due to the denaturation of some enzyme molecules, leaching of enzyme from the swollen polymer matrix, and degradation of polymer matrix [24]. Further, the high activity of immobilized enzyme at 50°C probably is a result of favoured adsorption of enzymes [25]. Lower activity of the immobilized enzyme has been observed during these assays as compared to the free enzyme. It might be due to decreased affinity of the enzyme for the substrate caused by internal diffusion of the immobilized enzyme [26].


The dependence of the rate constant on temperature of an enzyme catalyzed reaction can be represented by the Arrhenius equation [27]. For many reactions, the

values are in the range of 50–100 KJ mol −1 . But in the case of enzyme catalyzed reactions, the values are generally lower than those of non-enzyme-catalyzed reactions. The observed activation energies for free and immobilized enzyme were 54 and 14.44 KJ mol −1 , respectively. Similar results were obtained in case of aminoacylases immobilized by alkylation with iodoacetyl cellulose [28]. The values of for the immobilized enzyme are smaller than that for the free enzyme, implying that the immobilized enzymes are less sensitive [29].

The rates of thermal inactivation of the free and immobilized β-glucosidase were studied in the temperature range of 30–60°C and 50–80°C for 0–120 min (Figures 3(a) and 3(b)). The results indicated that the free β-glucosidase was fairly stable at the temperature range from 50 to 60°C while the immobilized β-glucosidase was fairly stable at 60–80°C. The above results suggest that the alginate matrix preserved the structure of the enzyme after immobilization process and it protected the enzyme from conformational changes caused by effects of temperature. The activity of immobilized enzyme decreased slowly and still retained 58% of its residual activity at 80°C. Similar results were obtained in immobilized araujiain without any significant loss in its activity at the studied temperatures [21]. The thermal stability of immobilized β-glucosidase increased considerably as a result of immobilization in the sodium alginate beads. This is because of immobilization and cross-linking which provided a more rigid external backbone for enzyme molecules. As a result, the effect of higher temperature in breaking the interactions that were responsible for the proper globular, catalytic active structure became less prominent, thus increasing the thermal stability of the immobilized enzyme [30].


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Watch the video: Immobilized Enzymes I. Enzyme specificity. Immobilization method. Carrier binding. Cross Linking (February 2023).