Properties of an amylase from thermophilic Bacillus SP

Abstract

α-Amylase production by thermophilic Bacillus sp strain SMIA-2 cultivated in liquid cultures containing soluble starch as a carbon source and supplemented with 0.05% whey protein and 0.2% peptone reached a maximum activity at 32 h, with levels of 37 U/mL. Studies on the amylase characterization revealed that the optimum temperature of this enzyme was 90°C. The enzyme was stable for 1 h at temperatures ranging from 40-50°C while at 90°C, 66% of its maximum activity was lost. However, in the presence of 5 mM CaCl2, the enzyme was stable at 90°C for 30 min and retained about 58% residual activity after 1 h. The optimum pH of the enzyme was found to be 8.5. After incubation of enzyme for 2 h at pH 9.5 and 11.0 was observed a decrease of about 6.3% and 16.5% of its original activity. At pH 6.0 the enzyme lost about 36% of its original activity. The enzyme was strongly inhibited by Co(2+), Cu(2+) and Ba(2+), but less affected by Mg(2+), Na(+) and K(+). In the presence of 2.0 M NaCl, 63% of amylase activity was retained after 2 h incubation at 45°C. The amylase exhibited more than 70% activity when incubated for 1 h at 50°C with sodium dodecyl sulphate. However, very little residual activity was obtained with sodium hypochlorite and with hydrogen peroxide the enzyme was completely inhibited. The compatibility of Bacillus sp SMIA-2 amylase with certain commercial detergents was shown to be good as the enzyme retained 86%, 85% and 75% of its activity after 20 min incubation at 50°C in the presence of the detergent brands Omo(®), Campeiro(®) and Tide(®), respectively.

Keywords: Bacillus sp; Thermophilic bacterium; α-Amylase.

Figure 1

Time course of α-amylase production by Bacillus sp strain SMIA-2 grown at 50°C on soluble starch (0.5%) in shake flasks. Results represent the means of three separate experiments, and bars indicated ± 1 standard deviation. Absence of bars indicates that errors were smaller than symbols.

Figure 2

Optimum pH (□) and stability (■) of α-amylase produced by Bacillus sp strain SMIA-2 grown at 50°C for 48 h. Relative activity is expressed as a percentage of the maximum (100% of enzyme activity = 36.1U/mL).

Figure 3

Optimum temperature (■) and stability temperature (□) of α-amylase produced by Bacillus sp strain SMIA-2 grown at 50°C for 48 h. Relative activity is expressed as a percentage of the maximum. 100% of enzyme activity = 36.4 U/mL.

Figure 4

Termostability of amylase at 90°C in the presence (■) or absence of Ca²⁺ (□). 100% of enzyme activity = 50.5 U/mL.

Figure 5

Effect of metal ions on amylase activity. The activity is expressed as a percentage of the activity level in the absence of metal ion. 100% of enzyme activity = 40.62 U/mL.

Figure 6

Effect of NaCl concentration on α-amylase produced by Bacillus sp strain SMIA-2 grown at 50°C for 24 h. Relative activity is expressed as a percentage of the maximum. 100% of enzyme activity = 34.8 U/mL.

Figure 7

Effect of inhibitors and oxidizing agents on α-amylase activity. The activity is expressed as a percentage of the activity level in the absence of inhibitors and oxidizing agents. 100% of enzyme activity = 31.9 U/mL.

Figure 8

Compatibility of α-amylase activity from Bacillus sp. SMIA-2 with commercial detergents (♦ Ariel^®, ■ Biz^®, Δ Cheer^®, x Tide^®, ▅ Campeiro®, ● Omo®). The activity is expressed as a percentage of the activity level in the absence of detergents (100% of enzyme activity = 47 U/mL).