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Electron density maps from the X-ray crystal structure of TAA MOD . Observed electron density (2 F o – F c ; light blue mesh; contoured at 1.5 SD) and
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The thermostability properties of TAA were investigated by chemically modifying carboxyl groups on the surface of the enzyme with AMEs. The TAA(MOD) exhibited a 200% improvement in starch-hydrolyzing productivity at 60 degrees C. By studying the kinetic, thermodynamic and biophysical properties, we found that TAA(MOD) had formed a thermostable, MG...
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... was not good enough for atomic modelling of the modifications. The modifying groups extend into the bulk solvent and they are poorly ordered, having no interactions with the remainder of the protein. There are two exceptions to this: D282 shows potential density (albeit discontinuous) for almost the whole of the arginine methylester modification (Fig. 5A) and sig- nificant electron density is observed near the carboxylate of D206 (Fig. 5B). The ordering of the modified D282 is prob- ably due to crystal packing, where the modifying group packs against a symmetry-related protein molecule. The density near D206 is intriguing, as this residue is in the active site of the enzyme. Although ...
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... into the bulk solvent and they are poorly ordered, having no interactions with the remainder of the protein. There are two exceptions to this: D282 shows potential density (albeit discontinuous) for almost the whole of the arginine methylester modification (Fig. 5A) and sig- nificant electron density is observed near the carboxylate of D206 (Fig. 5B). The ordering of the modified D282 is prob- ably due to crystal packing, where the modifying group packs against a symmetry-related protein molecule. The density near D206 is intriguing, as this residue is in the active site of the enzyme. Although the density is strong, it cannot be readily modelled in an unambiguous ...
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... MOD , k cat , 2240 + 142 min 21 ). Supplementary Fig. S5 shows the Michaelis -Menten plots. The X-ray structure of TAA MOD showed that the active-site residue, D206, was modified (Fig. 5B). ...
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... MOD , k cat , 2240 + 142 min 21 ). Supplementary Fig. S5 shows the Michaelis -Menten plots. The X-ray structure of TAA MOD showed that the active-site residue, D206, was modified (Fig. 5B). Comparison of peak heights in the electron density map of the modification suggests that 50% of the protein in the protein crystal was modified at this site. This is consistent with the loss of activity observed for TAA MOD . At 408C, K m of TAA UM and TAA MOD was 0.18 + 0.04% and 0.13 + 0.02% (w/v), respectively. Although no change ...
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Networks and clusters of intramolecular interactions, as well as their "communication" across the three-dimensional architecture have a prominent role in determining protein stability and function. Special attention has been dedicated to their role in thermal adaptation. In the present contribution, seven previously experimentally characterized mut...
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... To obtain further insights into the stability of horseradish peroxidase immobilized on the membrane, their thermodynamic activation parameters were determined at 25 • C. Relative to native HRP, the immobilized HRP on the PAN/PVdF nanofibrous membrane variant was stabilized enthalpically. A higher ∆H # when the process of horseradish peroxidase deactivation occurs implies that more bonds need to be broken to reach the partially unfolded transition state [34]. The enthalpy values ∆H # and the entropy ∆S # are lower for HRP nanofibrous membrane than the values ∆H # for native horseradish peroxidase (Table 4). ...
... The higher the ∆G # d is, the more stable the enzyme [27,35]. The increase in the stability of horseradish peroxidase immobilized on the membrane relative to native horseradish peroxidase is accompanied by a decrease of ∆H # and ∆S # values according to an enthalpy-entropy [34]. The thermodynamic parameters were shown and allowed to explain the thermal stabilization of immobilized horseradish peroxidase on the nanofibrous membrane, resulting from a decrease in entropy when the enzyme deactivation process does not occur. ...
Chlorophenol compounds pose a health risk to many organisms due to their toxicity. The present paper presents the estimation of the activation and deactivation energies and the optimum temperatures of 2,4-dichlorophenol degradation by horseradish peroxidase (HRP). The activities of horseradish peroxidase depending on temperature were analyzed. In a mathematical model, describing 2,4-dichlorophenol degradation by HRP was assumed that both the 2,4-dichlorophenol degradation and the deactivation of HRP were first-order reactions by the enzyme concentration. The parameters of the optimum temperatures Topt, the activation energies Er, and the deactivation energies Ed in the process of 2,4-dichlorophenol degradation by HRP immobilized on a modified nanofibrous membrane were determined kd and t(1/2)were determined for HRP immobilized at temperatures in the range of 25 °C to 75 °C. Likewise, thermodynamic parameters such as the change in the enthalpy ∆H#,change in entropy ∆S#, the change in Gibbs free energy ∆G# for native HPR and the change in the enthalpy ∆Hd#, change in entropy ∆Sd#, and the change in Gibbs free energy ∆Gd# for deactivated HRP were determined at 25 °C.
... The samples were excited at 380 nm and the emission spectra was recorded from 430 to 600 nm using a Jasco spectrofluorometer (FP 8200). The slit widths for both excitation and emission monochromators were set at 5 nm, and the scan speed was set at 100 nm/min [48]. ...
... The slit widths for both excitation and emission monochromators were set at 2.5 nm, and the scan speed was set at 100 nm/min. Acrylamide was used at a concentration range of 0-0.1 M. Thereafter, the Stern-Volmer plots for the unmodified and modified enzyme samples were plotted [48]. ...
... ANS fluorescence is primarily used for determining the hydrophobicity and solvent accessibility of binding sites on proteins. ANS is believed to bind to the cationic amino acid side chains of proteins, causing an increase in its fluorescence intensity accompanied by a blue shift in the emission maximum [48,65]. As can be observed in Fig. 10B, in its unbound state, ANS showed negligible fluorescence and maximum emission near 515-520 nm. ...
The present study describes the chemical modification of α-amylase using succinic anhydride (SA), phthalic anhydride (PA) and a novel modifier viz. 2-octenyl succinic anhydride (2-OSA). SA-, PA- and 2-OSA-α-amylases displayed a 50%, 91% and 46% increase in stability at pH 9, respectively; as compared to unmodified α-amylase. PA-α-amylase showed a significant increase in Ea and ΔHa#, and a concomitant decrease in ΔSa#. The modified α-amylases exhibited improved thermostability as reflected by significant reductions in Kd and ΔSd#, and increments in t1/2, D-, Ed, ΔHd# and ΔGd# values. The modified α-amylases displayed variable stabilities in the presence of different surfactants, inhibitors, metal ions and organic solvents. Interestingly, the chemical modification was found to confer resistance against inactivation by Hg²⁺ on α-amylase. The conformational changes in modified α-amylases were investigated using intrinsic tryptophan fluorescence, ANS (extrinsic) tryptophan fluorescence, and dynamic fluorescence quenching. Both intrinsic and extrinsic tryptophan fluorescence spectra showed increased fluorescence intensity for the modified α-amylases. Chemical modification was found to induce a certain degree of structural rigidity to α-amylase, as shown by dynamic fluorescence quenching. Analysis of the CD spectra by the K2d method using the DichroWeb online tool indicated evident changes in the α-helix, β-sheet and random coil fractions of the α-amylase secondary structure, following chemical modification using anhydrides. PA-α-amylase exhibited the highest productivity in terms of hydrolysis of starch at 60 °C over a period of 5 h indicating potential in varied biotechnological applications.
... After five cycles, the number of α-helices and β-turns in α-amylase decreased to 27.1% and 18.8%, respectively, while the percentage of β-sheets increased to 22% (nearly 2-fold). Based on the structural characteristics of α-amylase, the α-helices and β-turns played an important role in keeping catalysis [36]. As for the α-amylase/λ-carrageenan complexes (10:1), the initial secondary structure of αamylase did not change. ...
... Similar changes in heat-treated α-amylase indicated denaturing conditions [37]. According to the molecular characteristics of α-amylase, the secondary structure of the α-helix played an important role in maintaining catalysis [36]. The binding between α-amylase and λ-carrageenan might have exposed the catalytic domain to the external electric field, which became less protective against enzyme inactivation. ...
... After five cycles, the number of α-helices and β-turns in α-amylase decreased to 27.1% and 18.8%, respectively, while the percentage of β-sheets increased to 22% (nearly 2-fold). Based on the structural characteristics of α-amylase, the α-helices and β-turns played an important role in keeping catalysis [36]. As for the α-amylase/λ-carrageenan complexes (10:1), the initial secondary structure of α-amylase did not change. ...
Pulsed electric field (PEF) is an effective way to modulate the structure and activity of enzymes; however, the dynamic changes in enzyme structure during this process, especially the intermediate state, remain unclear. In this study, the molten globule (MG) state of α-amylase under PEF processing was investigated using intrinsic fluorescence, surface hydrophobicity, circular dichroism, etc. Meanwhile, the influence of coexisting carrageenan on the structural transition of α-amylase during PEF processing was evaluated. When the electric field strength was 20 kV/cm, α-amylase showed the unique characteristics of an MG state, which retained the secondary structure, changed the tertiary structure, and increased surface hydrophobicity (from 240 to 640). The addition of carrageenan effectively protected the enzyme activity of α-amylase during PEF treatment. When the mixed ratio of α-amylase to carrageenan was 10:1, they formed electrostatic complexes with a size of ~20 nm, and carrageenan inhibited the increase in surface hydrophobicity (<600) and aggregation (<40 nm) of α-amylase after five cycles of PEF treatment. This work clarifies the influence of co-existing polysaccharides on the intermediate state of proteins during PEF treatment and provides a strategy to modulate protein structure by adding polysaccharides during food processing.
... Therefore, the amount of reaction product at the end of an extended period of time is dependent upon the irreversible inactivation of enzyme due to thermal unfolding and/or substrate/product inhibition. In this way, different forms of an enzyme, such as native vs. modified, soluble vs. immobilized [9,[14][15][16] can be evaluated and more efficient enzymes can be identified and compared across studies. Moreover, productivity can be maximized in the presence of an additive [17] or by varying other reaction conditions, such as ionic strength, pH, and [S] and [E] concentrations [14]. ...
... Following 5 h at 60 • C, the modified enzyme hydrolyzed nearly twice the quantity of starch as compared with the unmodified enzyme. Although the modified α-amylase possessed only 60% of the intrinsic activity (k cat ) of the unmodified enzyme, the higher productivity attained by the modified enzyme was due to its relatively high thermostability over the duration of the hydrolysis reaction [16]. Productivity curves for alkaline metalloprotease, α-amylase (left), and AMP (right). ...
... Sodium acetate/acetic acid buffer (50 mM, pH 5) and enzyme (0.32 µg mL −1 ) were added to the starch solution (6% w/v), to initiate the reaction. Open symbols: Unmodified α-amylase; filled symbols: Modified α-amylase (reproduced from [16] with permission from Oxford University Press). Protein hydrolysis by native, EDTA-treated, and Ca 2+ -treated alkaline metalloproteases (AMP) at different temperatures. ...
Kinetic productivity analysis is critical to the characterization of enzyme catalytic perfor- mance and capacity. However, productivity analysis has been largely overlooked in the published literature. Less than 0.01% of studies which report on enzyme characterization present productivity analysis, despite the fact that this is the only measurement method that provides a reliable indicator of potential commercial utility. Here, we argue that reporting productivity data involving native, modified, and immobilized enzymes under different reaction conditions will be of immense value in optimizing enzymatic processes, with a view to accelerating biotechnological applications. With the use of examples from wide-ranging studies, we demonstrate that productivity is a measure of critical importance to the translational and commercial use of enzymes and processes that employ them. We conclude the review by suggesting steps to maximize the productivity of enzyme catalyzed reactions.
... Identifying those unique structural features that influence the stability in a thermally adapted set of homologous proteins has been a topical area of research with the view to defy activity-stability trade-off [13,14] in enzymes. The use of rational design [16] or chemical modification [13,17,18] has been employed to elucidate the structure-function-stability relationships in enzymes and to enhance their stability to suit various biotechnological applications [19]. Understanding the role of the structural features implicated in the stability of enzymes is crucial for designing thermostable chitinases for various biotechnological applications. ...
Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been
examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a “weak link” from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop “hot spots” for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure–stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions
... Siddiqui et al reported that the modification of carboxyl groups of TAA by L-arginine methyl ester dihydrochloride may improve the hydrolytic activity of alpha-amylase at 60°C. 73 Water is crucial for alpha-amylase activity; on the other hand, water can lead to denaturation of the enzyme. some stabilizing agents such as sugars or polyols can be added to alpha-amylase in order to change water structure and improve hydrophobic interaction strength in the enzyme structure. ...
Alpha-amylase reputes for starch modification by breaking of 1-4 glycosidic bands and is
widely applied in different industrial sectors. Microorganisms express unique alpha-amylases
with thermostable and halotolerant characteristics dependent on the microorganism’s intrinsic
features. Likewise, genetic engineering methods are applied to produce enzymes with higher
stability in contrast to wild types. As there are widespread application of α-amylase in industry,
optimization methods like RSM are used to improve the production of the enzyme ex vivo.
This study aimed to review the latest researches on the production improvement and stability
of α-amylase.
... Siddiqui et al reported that the modification of carboxyl groups of TAA by L-arginine methyl ester dihydrochloride may improve the hydrolytic activity of alpha-amylase at 60°C. 73 Water is crucial for alpha-amylase activity; on the other hand, water can lead to denaturation of the enzyme. some stabilizing agents such as sugars or polyols can be added to alpha-amylase in order to change water structure and improve hydrophobic interaction strength in the enzyme structure. ...
Alpha-amylase reputes for starch modification by breaking of 1-4 glycosidic bands and is widely applied in different industrial sectors. Microorganisms express unique alpha-amylases with thermostable and halotolerant characteristics dependent on the microorganism’s intrinsic features. Likewise, genetic engineering methods are applied to produce enzymes with higher stability in contrast to wild types. As there are widespread application of α-amylase in industry, optimization methods like RSM are used to improve the production of the enzyme ex vivo. This study aimed to review the latest researches on the production improvement and stability of α-amylase.
... No significant difference between the spectra can be observed, indicating that A4 dissolved in water does not perturb the conformation of α-glucosidase. To indicate the stabilization of α-glucosidase, the conformational flexibility was evaluated by DFQ measurements using acrylamide as a quencher for the Tyr/Trp residues [24]. Figure 8b shows the Stern-Volmer plots in the presence and absence of 50 mM A4 as a function of acrylamide concentration. ...
Activation and stabilization of enzymes is an important issue in their industrial application. We recently reported that synthetic betaines, derived from cellular metabolites, structure-dependently increased the activity and stability of various enzymes including hydrolases, oxidases, and synthetases simply by mixing them into the reaction buffer. In this report, we focus on amine N-oxides, which are similarly important metabolites in cells with a highly polarized N-oxide bond, and investigate their enzyme stabilization and activation behavior. It was revealed that synthetic amine N-oxides structure-dependently activate α-glucosidase-catalyzed hydrolysis reactions similarly to betaines. The subsequent comparison of the kinetic parameters, the optimal concentration range for activation, and the maximal activity, suggested that amine N-oxides facilitate hydrolysis reactions via the same mechanism as betaines, because no differences were confirmed. However, the enzyme stabilization effect of amine N-oxides was slightly superior to that of betaines and the temporal stability of the enzyme in aqueous solutions was higher in the low amine N-oxide concentration range. The rheological properties, CD spectra, and dynamic fluorescence quenching experiments suggested that the suppression of unfavorable conformational perturbation was related to the difference in the hydration environments provided by the surrounding water molecules. Thus, we clarified that amine N-oxides facilitate enzyme reactions as a result of their similarity to betaines and provide a superior stabilizing effect for enzymes. Amine N-oxides show potential for application in enzyme storage and long-term reactions.
... The approaches of modifications can further be explored for the enzyme like cellulase to overcome activity/stability trade-off in specific situations and conditions. Being frequently affected by substrate/product inhibition or enzyme unfolding, productivity of an enzyme reflects its performance capability at stipulated period of time under typical conditions (Siddiqui et al., 2010;Cavicchioli et al., 2011). Siddiqui et al. (2009) further verified that reducing inhibition from uncompetitive substrate using chemical modification can help in improving productivity at low temperature, which can be a valuable addition for commercial and/or industrial processes with high substrate concentrations. ...
Cold adapted bacteria undertake array of adaptive strategies and remain active with an active temperature range of 0–40 °C and activity in the acidic to neutral pH range (pH 4.5–7.0) to withstand in extreme environmental conditions and to retain metabolic and enzymatic activities. In soil, water or food, they were reported to comprise higher population as compared to mesophilic and thermophilic population (can make up to 86% of the total population). The present work critically discusses a detailed account of different strategies of cold adaptations in bacteria at cellular (viz. regulation of cell membrane fluidity through lipidcomposition, carotenoids production, cryoprotectants) and molecular level (differential gene expression, chaperones, proline isomerisation, antifreeze proteins, proteins and enzymes), cold-active cellulases from psychrophiles, their production strategies, the factors affecting and their properties and cold-active enzyme activities for different applications with special focus on cold-active cellulases. Due to more flexibility in protein structure and related higher binding capacity with the substrates, cold enzymes have more catalytic activities (around 10 times more than amesophilic enzyme) at cold temperatures. Attempt is made to critically analyze the practical relevance and significance of these aspects to help explore future directions of research related to scope of versatile applications of cold active enzyme.
... where v is the speed of the temperature gradient, C p is the excess heat capacity at a given temperature, Q t is the total heat evolved and Q is heat evolved at a given temperature 4,25,28,48 . The unfolding rates were then used to plot Arrhenius graphs and used as assistance in further analysis. ...
Protein stability is a widely studied topic, there are still aspects however that need addressing. In this paper we examined the effects of multiple proline substitutions into loop regions of the kinetically stable proteinase K-like serine protease VPR, using the thermostable structural homologue AQUI as a template. Four locations for proline substitutions were chosen to imitate the structure of AQUI. Variants were produced and characterized using differential scanning calorimetry (DSC), circular dichroism (CD), steady state fluorescence, acrylamide fluorescence quenching and thermal inactivation experiments. The final product VPRΔC_N3P/I5P/N238P/T265P was greatly stabilized which was achieved without any noticeable detrimental effects to the catalytic efficiency of the enzyme. This stabilization seems to be derived from the conformation restrictive properties of the proline residue in its ability to act as an anchor point and strengthen pre-existing interactions within the protein and allowing for these interactions to prevail when thermal energy is applied to the system. In addition, the results underline the importance of the synergy between distant local protein motions needed to result in stabilizing effects and thus giving an insight into the nature of the stability of VPR, its unfolding landscape and how proline residues can infer kinetic stability onto protein structures.
