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Macromolecular Crowding Enhances Catalytic Efficiency and Stability of α-Amylase.

Yadav JK - ISRN Biotechnol (2012)

Bottom Line: Based on the results it is suggested that the macromolecular crowding significantly improves the catalytic efficiency of the enzyme with marginal change in the structure.Out of four cosolvents examined, trehalose was found to be the most effective in consistently enhancing thermal stability of the enzyme.Moreover, the relative effectiveness of the above cosolvents was found to be dependent on their concentration used.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570 020, India ; Max-Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany.

ABSTRACT
In the present study an attempt was made to investigate the macromolecular crowding effect on functional attributes of α-amylase. High concentrations of sugar based cosolvents, (e.g., trehalose, sucrose, sorbitol, and glycerol) were used to mimic the macromolecular crowding environment (of cellular milieu) under in vitro conditions. To assess the effect of macromolecular crowding, the activity and structural properties of the enzyme were evaluated in the presence of different concentrations of the above cosolvents. Based on the results it is suggested that the macromolecular crowding significantly improves the catalytic efficiency of the enzyme with marginal change in the structure. Out of four cosolvents examined, trehalose was found to be the most effective in consistently enhancing thermal stability of the enzyme. Moreover, the relative effectiveness of the above cosolvents was found to be dependent on their concentration used.

No MeSH data available.


Related in: MedlinePlus

Far-UV CD spectra of α-amylase in the presence of cosolvents. Residual molar ellipticity was measured from 200 to 260 nm at 25°C in the absence (a) and presence of 20%  (w/v) glycerol (b), sorbitol (c), sucrose (d) and trehalose (e). Each spectrum represents an accumulation of five independent scans.
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fig3: Far-UV CD spectra of α-amylase in the presence of cosolvents. Residual molar ellipticity was measured from 200 to 260 nm at 25°C in the absence (a) and presence of 20%  (w/v) glycerol (b), sorbitol (c), sucrose (d) and trehalose (e). Each spectrum represents an accumulation of five independent scans.

Mentions: After having observed the effect of cosolvents on the enzyme function and stability, a curiosity developed to investigate the biophysical properties of the enzyme in the presence of cosolvents. The structural analyses of the enzyme in the presence of cosolvents were performed by using far UV-CD and intrinsic fluorescence measurement at ambient temperature. The data from far UV-CD measurement indicate a minor change in the ellipticity in the presence of 20% (w/v), as shown in Figure 3. A slight negative increase in the peaks at 222 nm and 216 nm indicates a minor change in α-helical and β-sheet components. The intrinsic fluorescence intensity of the enzyme was found to be slightly decreased in the presence of all the cosolvents as shown in Figure 4. In addition, an small shift in the emission maxima (λmax) was also observed at least in the presence of sucrose and sorbitol. The λmax is an excellent parameter to monitor the polarity of tryptophan microenvironment in enzyme molecule and it is sensitive to change in protein conformation [22]. The λmax of α-amylase in buffer was found to be 340 nm and it was found to be 336 nm and 337 nm in the presence of 40% (w/v) sucrose and sorbitol, respectively (Figure 5). A reduction in intrinsic fluorescence intensity and shifting of λmax towards lower wavelength indicate that the aromatic residues are being relocated in more hydrophobic environment [23]. Together with far-UV-CD and fluorescence data it is clear that the presence of cosolvents makes some changes in the enzyme structure which may be accountable for tremendous effect on activity and thermal stability.


Macromolecular Crowding Enhances Catalytic Efficiency and Stability of α-Amylase.

Yadav JK - ISRN Biotechnol (2012)

Far-UV CD spectra of α-amylase in the presence of cosolvents. Residual molar ellipticity was measured from 200 to 260 nm at 25°C in the absence (a) and presence of 20%  (w/v) glycerol (b), sorbitol (c), sucrose (d) and trehalose (e). Each spectrum represents an accumulation of five independent scans.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4403623&req=5

fig3: Far-UV CD spectra of α-amylase in the presence of cosolvents. Residual molar ellipticity was measured from 200 to 260 nm at 25°C in the absence (a) and presence of 20%  (w/v) glycerol (b), sorbitol (c), sucrose (d) and trehalose (e). Each spectrum represents an accumulation of five independent scans.
Mentions: After having observed the effect of cosolvents on the enzyme function and stability, a curiosity developed to investigate the biophysical properties of the enzyme in the presence of cosolvents. The structural analyses of the enzyme in the presence of cosolvents were performed by using far UV-CD and intrinsic fluorescence measurement at ambient temperature. The data from far UV-CD measurement indicate a minor change in the ellipticity in the presence of 20% (w/v), as shown in Figure 3. A slight negative increase in the peaks at 222 nm and 216 nm indicates a minor change in α-helical and β-sheet components. The intrinsic fluorescence intensity of the enzyme was found to be slightly decreased in the presence of all the cosolvents as shown in Figure 4. In addition, an small shift in the emission maxima (λmax) was also observed at least in the presence of sucrose and sorbitol. The λmax is an excellent parameter to monitor the polarity of tryptophan microenvironment in enzyme molecule and it is sensitive to change in protein conformation [22]. The λmax of α-amylase in buffer was found to be 340 nm and it was found to be 336 nm and 337 nm in the presence of 40% (w/v) sucrose and sorbitol, respectively (Figure 5). A reduction in intrinsic fluorescence intensity and shifting of λmax towards lower wavelength indicate that the aromatic residues are being relocated in more hydrophobic environment [23]. Together with far-UV-CD and fluorescence data it is clear that the presence of cosolvents makes some changes in the enzyme structure which may be accountable for tremendous effect on activity and thermal stability.

Bottom Line: Based on the results it is suggested that the macromolecular crowding significantly improves the catalytic efficiency of the enzyme with marginal change in the structure.Out of four cosolvents examined, trehalose was found to be the most effective in consistently enhancing thermal stability of the enzyme.Moreover, the relative effectiveness of the above cosolvents was found to be dependent on their concentration used.

View Article: PubMed Central - PubMed

Affiliation: Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570 020, India ; Max-Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany.

ABSTRACT
In the present study an attempt was made to investigate the macromolecular crowding effect on functional attributes of α-amylase. High concentrations of sugar based cosolvents, (e.g., trehalose, sucrose, sorbitol, and glycerol) were used to mimic the macromolecular crowding environment (of cellular milieu) under in vitro conditions. To assess the effect of macromolecular crowding, the activity and structural properties of the enzyme were evaluated in the presence of different concentrations of the above cosolvents. Based on the results it is suggested that the macromolecular crowding significantly improves the catalytic efficiency of the enzyme with marginal change in the structure. Out of four cosolvents examined, trehalose was found to be the most effective in consistently enhancing thermal stability of the enzyme. Moreover, the relative effectiveness of the above cosolvents was found to be dependent on their concentration used.

No MeSH data available.


Related in: MedlinePlus