Limits...
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

Effect of cosolvents on activity and optimum temperature of α-amylase. Figures 2(a), 2(b), 2(c), and 2(d) represent the activity profiles of the enzyme in the presence of glycerol, sorbitol, sucrose, and trehalose, respectively. The activity of the enzyme was measured at different temperatures and curves represented as the enzyme (a) in buffer only, (b) in 10%, (c) in 20%, (d), in 30%, and (e) in 40% (w/v) of respective cosolvents.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4403623&req=5

fig2: Effect of cosolvents on activity and optimum temperature of α-amylase. Figures 2(a), 2(b), 2(c), and 2(d) represent the activity profiles of the enzyme in the presence of glycerol, sorbitol, sucrose, and trehalose, respectively. The activity of the enzyme was measured at different temperatures and curves represented as the enzyme (a) in buffer only, (b) in 10%, (c) in 20%, (d), in 30%, and (e) in 40% (w/v) of respective cosolvents.

Mentions: Although the increase in thermal resistance of the enzyme appeared attractive, it was not surprising as the enhancement in the thermal stability of various enzymes and proteins in the presence of cosolvents is well documented previously [19–21]. However, the simultaneous observation of the cosolvent-mediated enhancement in the catalytic activity of the enzyme was unexpected. Mere presence of cosolvents significantly improved the catalytic efficiency of the enzyme. The cosolvent-induced enhancement in activity was further noticeable when the activity was measured at higher temperatures, as shown in Figure 2. Moreover, a shift in the temperature for optimum enzyme activity was also observed, at least, in the presence of sucrose and glycerol. As shown in Figure 2, the enzyme in buffer has optimum activity around 50°C. In the presence of cosolvents the enzyme activity was found to be higher at >50°C. The enhancement in the enzyme activity was found to dependent on the concentration of each cosolvent used, at least in the range of 10–40% (w/v). This finding could be of vital importance in the processes where the enzymatic activity is carried out at higher temperature (e.g., starch saccharification and liquefaction, temperature reaches up to 70–100°C). Further, such enhancement in the enzyme activity was found to be reversible. Table 1 lists the activity of the enzyme before treatment with cosolvents and after their removal from enzyme solution. After removal of cosolvents the enzyme activity was found to be nearly identical to the enzyme without any treatment.


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

Yadav JK - ISRN Biotechnol (2012)

Effect of cosolvents on activity and optimum temperature of α-amylase. Figures 2(a), 2(b), 2(c), and 2(d) represent the activity profiles of the enzyme in the presence of glycerol, sorbitol, sucrose, and trehalose, respectively. The activity of the enzyme was measured at different temperatures and curves represented as the enzyme (a) in buffer only, (b) in 10%, (c) in 20%, (d), in 30%, and (e) in 40% (w/v) of respective cosolvents.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Effect of cosolvents on activity and optimum temperature of α-amylase. Figures 2(a), 2(b), 2(c), and 2(d) represent the activity profiles of the enzyme in the presence of glycerol, sorbitol, sucrose, and trehalose, respectively. The activity of the enzyme was measured at different temperatures and curves represented as the enzyme (a) in buffer only, (b) in 10%, (c) in 20%, (d), in 30%, and (e) in 40% (w/v) of respective cosolvents.
Mentions: Although the increase in thermal resistance of the enzyme appeared attractive, it was not surprising as the enhancement in the thermal stability of various enzymes and proteins in the presence of cosolvents is well documented previously [19–21]. However, the simultaneous observation of the cosolvent-mediated enhancement in the catalytic activity of the enzyme was unexpected. Mere presence of cosolvents significantly improved the catalytic efficiency of the enzyme. The cosolvent-induced enhancement in activity was further noticeable when the activity was measured at higher temperatures, as shown in Figure 2. Moreover, a shift in the temperature for optimum enzyme activity was also observed, at least, in the presence of sucrose and glycerol. As shown in Figure 2, the enzyme in buffer has optimum activity around 50°C. In the presence of cosolvents the enzyme activity was found to be higher at >50°C. The enhancement in the enzyme activity was found to dependent on the concentration of each cosolvent used, at least in the range of 10–40% (w/v). This finding could be of vital importance in the processes where the enzymatic activity is carried out at higher temperature (e.g., starch saccharification and liquefaction, temperature reaches up to 70–100°C). Further, such enhancement in the enzyme activity was found to be reversible. Table 1 lists the activity of the enzyme before treatment with cosolvents and after their removal from enzyme solution. After removal of cosolvents the enzyme activity was found to be nearly identical to the enzyme without any treatment.

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