Thermal Stability Of Α-amylase Research Articles

Structural evidence for kinetic and thermal stability changes of α-amylase due to exposure to [emim][lactate] ionic liquid

Abstract Objectives α-amylases hydrolyze α-1,4 glycosidic bonds in starch. ILs used as co-solvent in different enzymatic reactions to improve activity, selectivity and stability of enzymes. In this study, fluorescence spectroscopy method was used to explain the effect of [emim][lactate] on kinetic and thermal stability of Aspergillus oryzae α-amylase. Methods Effect of different concentrations of [emim][lactate] on activity of α-amylases was determined. Kinetic parameters, optimum pH and temperature and thermal stability were determined and compared with absence of [emim][lactate]. Intrinsic fluorescence spectroscopy for Trp residues was performed for both presence and absence of [emim][lactate]. Results Activity of α-amylase decreases in presence of [emim][Lac]. Moreover, Km of α-amylase in the presence of [emim][lactate] increases while Vm decreased. Optimum temperature in presence of [emim][lactate] increases from 45 to 50 °C while optimum pH decreases from 9 to 7. Thermal stability of α-amylase in the presence of [emim][lactate] is similar to that in the absence of [emim][lactate] at 40 and 50 °C but decreases at 60 °C. Intrinsic fluorescence spectroscopy shows unfolding of native structure of α-amylase is dependent on [emim][lactate] concentration. Conclusions Presence of [emim][lactate] ionic liquid as co-solvent leads to structural unfolding of α-amylase and loss of its activity and thermal stability.

Increasing Stability of a-amylase Obtained from Bacillus subtilis ITBCCB148 by Immobilization with Chitosan

In this research, the immobilization of α-amylase from Bacillus subtilis ITBCCB148 by crosslinking method on chitosan matrix has been performed. This research aims to know the effect of immobilization on the thermal stability of α-amylase. The results showed that the native α-amylase has an optimum temperature of 65oC, KM = 1.6 mg mL-1 substrate, and Vmax = 39.7 µmol mL-1 min-1. The immobilized α-amylase has optimum temperature of 75oC, KM = 3.5 mg mL-1 substrate, and Vmax = 7.05 µmol mL-1 min-1. The residual activity of the native and immobilized enzyme on thermal stability test at 65oC for 80 minutes was 58% and 86.15%, respectively. The immobilized enzyme can be reused up to six repeated cycles.The thermodynamic data of native enzyme was t½ = 113.6 min, ki = 6.1x10-3 min-1, and ΔGi = 107.3 kJ mol-1, while the immobilized enzyme was t½ = 433.1 min, ki= 1.6x10-3 min-1, and ΔGi 111.1 kJ mol-1. Based on the decrease of ki, and the increase of ΔGi and half-life(t½) values, the immobilization of α-amylase with chitosan can increase the thermal stability of this enzyme.

Effect of additives on mesophilic α-amylase and its application in the desizing of cotton fabrics

An investigation was carried out on thermal stability of α-amylase. The influence of various additives (calcium acetate, sodium lactate, L-histidine, and water-soluble chitosan) on the stability of α-amylase was studied. Results showed the inactivation behavior of α-amylase with or without additives all followed the first-order kinetics. All additives (Ca2+, sodium lactate, L-histidine, and water-soluble chitosan) displayed good stabilizing effect on α-amylase lower than 80 °C, and only water-soluble chitosan had an efficient stabilizing effect on α-amylase when the treatment temperature exceeds 80 °C. All additives improved the catalytic activity of α-amylase at 70–90 °C, and the appearance of water-soluble chitosan increased the catalytic activity of α-amylase at 90 °C sharply. A desizing ratio of 68.42% was obtained by treating the cotton fabrics in the buffer solution at 100 °C without α-amylase. To obtain a desizing ratio exceed 95% when fabrics were treated at 100 °C for 10 min, the addition of water-soluble chitosan saves 2/3 α-amylase dosage. Moreover, water-soluble chitosan showed a further improvement in desizing effect than the additive of calcium acetate.

Effect of Heavy Atoms on the Thermal Stability of α-Amylase from Aspergillus oryzae

Currently, there are no versatile and established methods for improving stability of proteins. In an entirely different approach from conventional techniques such as mutagenesis, we attempted to enhance enzyme stability of α-amylase from Aspergillus oryzae using a heavy-atom derivatization technique. We evaluated changes in stability using differential scanning calorimetry (DSC). Candidate heavy atoms were identified using the Heavy-Atom Database System HATODAS, a Web-based tool designed to assist in heavy-atom derivatization of proteins for X-ray crystallography. The denaturation temperature of α-amylase derivatized with gadolinium (Gd) or samarium (Sm) ions increased by 6.2 or 5.7°C, respectively, compared to that of the native protein (60.6°C). The binding of six Gd ions was confirmed by X-ray crystallography of the enzyme at 1.5 Å resolution. DSC and dynamic light-scattering data revealed a correlation between stability and the aggregation state upon addition of Gd ions. These results show that HATODAS search is an effective tool for selecting heavy atoms for stabilization of this protein.

Thermal stability of α-amylase in aqueous cosolvent systems

The activity and thermal stability of alpha-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 +/- 2 degrees C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (Tm) app and activation energy (Ea) of alpha-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (Tm)app were 20 degrees C, 14 degrees C, 13 degrees C and 9 degrees C, respectively. The Ea of thermal denaturation of alpha-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents.