Stability Of Alpha-amylase Research Articles

Effect of N-linked glycosylation on secretion, activity, and stability of alpha-amylase from Aspergillus oryzae.

The effect of N-linked glycosylation on secretion, activity, and stability of alpha-amylase from Aspergillus oryzae grown as dispersed filaments was studied. In the presence of tunicamycin the fungus grew either as dispersed filaments or as one large pellet, whereas growth was as dispersed filaments in all control cultures. The presence of tunicamycin affected neither biomass, level of secreted alpha-amylase, nor total amount of secreted protein in cultures growing as dispersed filaments. In these cultures both glycosylated and nonglycosylated alpha-amylase appeared in the culture medium as well as in the cells, whereas in control cultures only the glycosylated form of alpha-amylase was found in the medium and in the cells. The presence of nonglycosylated alpha-amylase in the medium seemed to result from active secretion rather than from autolysis of the mycelium or extracellular deglycosylation. Deglycosylation with Endo H of crude alpha-amylase in culture filtrate did not affect its stability towards heat, acid pH, or proteolytic degradation.

Effect of polyols on fungal alpha-amylase thermostability

The influence of different polyols (ethylene glycol, glycerol, erythritol, xylitol, and sorbitol) on the thermostability of fungal alpha-amylase at 60°C has been studied. The results obtained show a stabilizing effect in the presence of polyols. In the case of 4 m sorbitol solution, the enzyme half-life is 2000-fold longer than in pure water. These polyols have been found as competitive inhibitors for alpha-amylase andtheir stabilizing effect has been correlated to their affinity constant except for sorbitol. The influence of two polyol isomers (arabitol and mannitol) on activity and stability of alpha-amylase has also been investigated.

Physico-chemical properties of Aspergillus flavus var. columnaris α-amylase

The best temperature for the preservation of A. flavus var. columnaris alpha-amylase was -5 degrees C followed by 5 degrees C. CaCl2 at 0.005 M had no effect on the activity in both temperatures. Repeated freezing (-5 degrees C) and thawing followed by freezing (-5 degrees C) had no effect on stability of alpha-amylase. On the other hand, 25 degrees C was the lowest preservation temperature without any effect on the stability on alpha-amylase. 0.005 M CaCl2 decreased the activity of alpha-amylase and reached a 100% inhibition at 35th day. The fungal alpha-amylase had an optimum temperature of 55 degrees C at pH 4.6, but had 60 degrees C in buffer containing 0.005 M CaCl2 and 50 degrees C in buffer containing 0.005 M Na2-EDTA. The addition of 0.01 M CaCl2 greatly increased the thermostability of alpha-amylase at 40, 45, 50, 55 and 60 degrees C for 30 min. Optimum pH for alpha-amylase only was 5, but in the presence of 0.01 M CaCl2 or Na2-EDTA 5.6. The enzyme only was stable for 4 h at 25 degrees C. Whereas addition of 0.01 M CaCl2 showed a loss of 4% compared to a 22% loss in the presence of 0.01 M Na2-EDTA after 4 h at 25 degrees C and 65% loss in the presence of 0.01 M CaCl2 together with 0.01 M Na2-EDTA in the beginning and a 100% loss after 4 h at 25 degrees C. The optimum temperature for the activity of alpha-amylase at pH 5 was 50 degrees C for the enzyme only but 55 degrees C in the presence of 0.01 M CaCl2. However, at pH 6 and 7 optimum temperature was 55 degrees C for the activity of the enzyme only or with 0.01 M CaCl2. The presence of 0.01 M CaCl2 at pH 5, 6 and 7 resulted in increase of enzyme activity at the temperatures above 50, 40 and 25 degrees C, respectively. However, 0.01 M CaCl2 at pH 5 and 6 resulted in decreasing enzyme activity at temperatures below 55 and 45 degrees C, respectively.