Highly thermostable and alkaline α-amylase from a halotolerant-alkaliphilic Bacillus sp. AB68

Abstract

An alkaliphilic and highly thermostable α-amylase producing Bacillus sp. was isolated from Van soda lake. Enzyme synthesis occurred at temperatures between 25°C and 40°C. Analysis of the enzyme by SDS-PAGE revealed a single band which was estimated to be 66 kDa. The enzyme was active in a broad temperature range, between 20°C and 90°C, with an optimum at 50°C; and maximum activity was at pH 10.5. The enzyme was almost completely stable up to 80°C with a remaining activity over 90% after 30 min pre-incubation. Thermostability was not increased in the presence of Ca(2+). An average of 75% and 60°C of remaining activity was observed when the enzyme was incubated between pH 5 and 9 for 1 h and for 2 h, respectively. The activity of the enzyme was inhibited by SDS and EDTA by 38% and 34%, respectively.

Keywords: Alkaliphilic; Bacillus sp; Enzyme; Thermostable; α-amylase.

Figure 1

Zymogram and SDS-PAGE analysis of Amylase. Lane A: Amylase was run on native-PAGE, then soaked in 10 g/L of soluble starch in 50 mM Glycine-NaOH pH 10.0 and incubated with shaking (60 rpm) at 50°C for 30 min. Activity zones on gel were then visualised with a solution containing 5 g/L KI and 0.5 g/L I₂.The sample was subjected to SDS-PAGE and the protein bands visualized with Coomasie Brillant Blue staining. Lane 1: 30 μL and Lane 2: 15 μL of Amylase. M: SDS-6H High Molecular Weights Standard Mixture (Sigma, Carbonic Anhydrase 29; Ovalbumin 45; Bovine Albumin 66; Phosphorylase 97.4; β-Galactosidase 116 and Myosine 205 kDa).

Figure 2

Effect of pH on the activity of Bacillus sp. AB68 Amylase. The reaction mixture contained 0.5 mL substrate (1% soluble starch) and 0.5 mL enzyme. The reaction mixture was incubated at 50°C for 30 min. The buffers were 100 mM Citrate-Phosphate (pH 4.0–6.0), Na-Phosphate (pH 6.5–8.0), Glycine-NaOH (pH 8.5–10.5) and Borax-NaOH (pH 11.0–13.0).

Figure 3

Effect of temperature on the activity of Bacillus sp. AB68 amylase. The reaction mixture contained 0.5 mL substrate (1% soluble starch in Glycine-NaOH buffer, pH 10.5) and 0.5 mL enzyme. The mixture was incubated for 30 min at temperatures from 20 to 100°C under standard enzyme assay conditions.

Figure 4

Effect of pH on the stability of Bacillus sp.AB68 Amylase. For determination of pH stability of amylase AB68, the enzyme was pre-incubated in buffers at 50°C for 1 (◆) and 2 hours (■). The buffers used were 100 mM Citrate-Phosphate (pH 4.0–6.0), 100 mM Na-Phosphate (pH 6.5–8.0), 100 mM Glycine-NaOH (pH 8.5–10.5), and 100 mM Borax-NaOH (pH 11.0–13.0).

Figure 5

Thermal stability of Bacillus sp. AB68 amylase. The enzyme was pre-incubated at temperatures from 20 to 100°C for 30 min (◆) and 60 min (■) at optimum pH, subsequently remaining activity (%) was determined under standard enzyme condition.

Figure 6

Effect of salt concentration on enzyme activity (•) and stability (■,▲) of Bacillus sp. AB68 amylase. The reaction mixture for enzyme activity contained 0.5 mL substrate (1% soluble starch in Glycine-NaOH buffer, pH 10.5) with NaCl and 0.5 mL enzyme. Enzyme stability was tested by pre incubating the enzyme at 50°C, pH 10.5 for 30 (■) and 60 min (▲).

Figure 7

Thin layer chromatography of enzyme products from Bacillus sp. AB68. N: 5 mL Untreated soluble starch 1% (w/v); G: 5 μL of Glucose 4% (w/v); M: 5 μL of Maltose, 4% (w/v); AB68: 5 μL of enzyme substrate mixture.