Stabilizing effect of various polyols on the native and the denatured states of glucoamylase

Abstract

Different spectral probes were employed to study the stabilizing effect of various polyols, such as, ethylene glycol (EG), glycerol (GLY), glucose (GLC) and trehalose (TRE) on the native (N), the acid-denatured (AD) and the thermal-denatured (TD) states of Aspergillus niger glucoamylase (GA). Polyols induced both secondary and tertiary structural changes in the AD state of enzyme as reflected from altered circular dichroism (CD), tryptophan (Trp), and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence characteristics. Thermodynamic analysis of the thermal denaturation curve of native GA suggested significant increase in enzyme stability in the presence of GLC, TRE, and GLY (in decreasing order) while EG destabilized it. Furthermore, CD and fluorescence characteristics of the TD state at 71°C in the presence of polyols showed greater effectiveness of both GLC and TRE in inducing native-like secondary and tertiary structures compared to GLY and EG.

Figure 1

Effect of various polyols, such as, glucose (GLC), trehalose (TRE), glycerol (GLY), and ethylene glycol (EG), on the far-UV CD spectra of the native and the acid-denatured GAs. Different line symbols represent native GA (thick curve), acid-denatured GA (thin curve), native GA + polyol (dashed curve), and acid-denatured GA + polyol (dotted curve). The spectra were recorded at 25°C using a protein concentration of 1.4 μM. Different polyol concentrations used were 2.6 M GLC, 1.3 M TRE, 8.0 M GLY, and 8.0 M EG.

Figure 2

Effect of various polyols, such as, glucose (GLC), trehalose (TRE), glycerol (GLY), and ethylene glycol (EG), on the tryptophan fluorescence spectra of the native and the acid-denatured GAs. Different line symbols represent native GA (thick curve), acid-denatured GA (thin curve), native GA + polyol (dashed curve), and acid-denatured GA + polyol (dotted curve). The spectra were recorded at 25°C using a protein concentration of 0.12 μM. Different polyol concentrations used were 2.6 M GLC, 1.3 M TRE, 8.0 M GLY, and 8.0 M EG.

Figure 3

ANS fluorescence spectra of the acid-denatured GA in the absence (thick curve) and the presence of 2.6 M GLC (dashed curve), 1.3 M TRE (dotted curve), 8 M GLY (dashed with one dot curve), and 8 M EG (dashed with two dots curve). The spectra were recorded at 25°C, using a protein concentration of 0.26 μM and ANS : protein molar ratio as 70 : 1.

Figure 4

Normalized thermal transition curves of the native GA in the absence (■) and the presence of 2.6 M GLC (▲), 1.3 M TRE (○), 8 M GLY (◊), and 8 M EG (▼) as monitored by MRE_222 nm measurements, using a protein concentration of 1.4 μM.

Figure 5

Far-UV CD spectra of the native (thick curve) and the thermal-denatured GAs in the absence (thin curve) and the presence of 2.6 M GLC (dashed curve), 1.3 M TRE (dotted curve), 8 M GLY (dashed with one dot curve), and 8 M EG (dashed with two dots curve). The spectra of the thermal-denatured GA were recorded after equilibrating the sample at 71°C for 6 min, using a protein concentration of 1.4 μM.

Figure 6

Tryptophan fluorescence spectra of the native (thick curve) and the thermal-denatured GAs in the absence (thin curve) and the presence of 2.6 M GLC (dashed curve), 1.3 M TRE (dotted curve), 8 M GLY (dashed with one dot curve) and 8 M EG (dashed with two dots curve). The spectra of thermal-denatured GA were recorded after equilibrating the sample at 71°C for 6 min, using a protein concentration of 0.12 μM.