Macromolecular Crowding Enhances Catalytic Efficiency and Stability of α-Amylase

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.

Figure 1

Effect of cosolvents on thermal stability of α-amylase. The enzyme solutions in the respective buffer or cosolvent solutions were incubated at 60°C for different period of time and residual activity was evaluated. The error bar represents the standard deviation.

Figure 2

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.

Figure 3

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.

Figure 4

Intrinsic fluorescence of α-amylase in the presence of cosolvents. Figures 4(a), 4(b), 4(c), and 4(d) represent the activity profiles of the enzyme in the presence of glycerol, sorbitol, sucrose, and trehalose, respectively. The curves are represented as (a) the enzyme in buffer, (b) in 20%, and (c) in 40% of respective cosolvents.

Figure 5

Effect of cosolvents on λ _max of α-amylase. The curves represent the changes in emission maxima of the enzyme in the presence of different concentrations of sucrose (a) and sorbitol (b).