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Partial Characterization of Extracellular ?-amylase from Three Bacillus Isolates

Arifa Nusrat
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh

Sabita Rezwana Rahman
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh

Abstract/ Executive Summary:

α-Amylase produced by three Bacillus isolates had been compared on the basis of the following criteria: heat and pH effects on activity and stability, effect of metal ions on enzyme activity and kinetic parameters. The culture filtrates obtained by growing the organisms in starch medium were fractionated by ammonium sulphate precipitation technique, and the highest enzyme activities were recovered from 70% saturation fraction. The enzyme from Bacillus amyloliquefaciens showed optimum activity at 60°C, while B. subtilis and another Bacillus isolate at lower (55°C) temperature. The pH optima of the enzymes from all sources were between 6.5 and 7.0 with an optimum reaction time of 10 to 15 min. α-Amylases were moderately thermostable exhibiting almost full activities at 50°C for at least 20 min. The enzyme from all sources showed stability over a wide range of pH (4.0-8.5). The apparent Km values on soluble starch varied between 1.6 mg/ml in case of B. amyloliquefaciens and 2.5 mg/ml in case of B. subtilis. Metal ions like Mg²⁺ and Mn²⁺ seemed to have positive influence on the enzyme activities of B. subtilis and Bacillus sp. The enzyme activities from three isolates were strongly inhibited by Cu²⁺, Hg²⁺ and Zn²⁺.

Keywords: Extracellular ?-amylase, Bacillus spp., Enzyme characterization

Location: Department of Microbiology, University of Dhaka

Start Date:

End Date:

Program Area: Knowledge Management

Commodity: Fermenters

Objectives:

The present article reports the partial purification and characterization of α-amylases of the three Bacillus isolates.

Methodology:

B. subtilis, B. amyloliquefaciens and another Bacillus isolate were grown in the medium containing 1.0% starch, 0.5% peptone, 0.5% corn steep liquor, 0.8% (NH4)2SO4, 0.2% MgSO₄.7H₂O, 0.05% CaCl₂.2H₂O, 1.4% K₂HPO₄and 0.6% KH₂PO₄ with an initial pH 7.06. Shake-flask cultures were carried out in 250-ml Erlenmeyer flasks at 37°C for at least 72 h with continuous shaking (150 rpm) in an orbital shaker incubator. Extracellular α-amylases were removed by centrifugation and the enzyme activity was measured in the cell-free supernatant essentially. The reducing sugar released was determined by the method of Miller. One unit (U) of enzyme activity was defined in all cases as the amount of enzyme releasing 1 μmol of glucose or glucose equivalents from the substrate per min under the assay conditions. For partial purification of the enzyme, the soluble protein contents of the culture supernatants were precipitated by stepwise addition of solid ammonium sulphate to 100% saturation. For temperature optima, the partially purified enzymes were assayed at different temperatures between 30 and 80°C with standard incubation time. The activity vs. pH profile was determined according to standard assay procedure at various pH values using 0.05 M citrate buffer (pH 4.0-6.0), 0.05 M phosphate buffer (pH 6.5-8.5) and 0.05 M Tris-HCl buffer (pH 9.0-10.0). Thermostability experiments were performed by incubating the enzyme preparation in 0.5 M phosphate buffer (pH 7.0) at various temperatures (4-100°) for 20 min. For determination of pH stability of α-amylase, the enzyme preparation in various pH buffers were incubated at 4°C for 24 h, and thereafter the residual activities were measured by standard procedure. For kinetic analysis, the α-amylase activity was assayed using various substrate (soluble starch) concentrations (0.5-6.0 mg/ml) under standard assay conditions. The enzyme kinetic parameters, namely K_m and V_max, were derived using double reciprocal plot. The apparent K_m of α-amylase was expressed as mg/ml and the V_max of the enzyme as U/mg protein. The effect of various metal ions on the enzyme activity was measured by incubation the enzyme preparation at 4°C and pH 7.0 for 1 h in the presence of various chemicals at 5 mM concentration. The residual activities were measured by standard procedure. α-Amylase produced by the three Bacillus isolates was partially purified by ammonium sulphate fractionation. Total soluble protein as well as α-amylase activity were obtained in 70% ammonium sulphate saturation fraction. Similar result was also reported for α-amylase produced by Streptomyces chattanoogensis. The enzyme was assayed at different temperatures (30-80°C), pH (4.0- 10.0) and reaction time (5-60 min). The optimum temperatures for α-amylase were 60°C in case of B. amyloliquefaciens and 55°C in case of B. subtilis and Bacillus sp. The enzyme activity drastically decreased with the increase of temperature from their optimum temperatures that indicates a rapid inactivation of enzyme. These findings were in accordance to that reported by other investigators on α-amylases from different Bacillus strains. In another study, the optimum temperature of α-amylase from yeast, Saccharomyces cerevisiae, was found to be 50ºC11. All the assayed activity had an optimum near neural pH (6.5-7.0). The maximum α-amylase activity of B. subtitis and Bacillus sp. was obtained after 15 min incubation, while the optimum reaction time for the enzyme from B. amyloliquefaciens was shorter (10 min).

Source of Information: Bangladesh J Microbiol, Volume 25, Number 1, June 2008, pp 76-78

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In view of the results obtained in this study, Bacillus isolates appear to be good sources of α-amylase with regard to temperature and pH effects. Little differences in pH stability were found among the α-amylases produced by the three isolates. However, a remarkable thermal stability of the enzyme was found for B. amyloliquefaciens. Metal ions such as Ca²⁺, Mn²⁺ and Fe²⁺ had little or no influence on the enzymes from B. subtilis and Bacillus sp., but they exhibited strong inhibitory effect on the enzyme from B. amyloliquefaciens.

Knowledge Management: Journal