Purification and Characterization of Thermostable α-Amylase from Soil Bacterium Bacillus sp.

BACKGROUND: Amylases are used in several industrial and biotechnological sectors, including those producing textiles, detergents, paper and bakery products. OBJECTIVE: This study aimed to purify an industrially important α-amylase from Bacillus sp. For this purpose, a single and rapid α-amylase purification was performed using the starch affinity method. METHODS: Characterization of the purified enzyme was determined by investigating temperature, pH stability, detergents, and metal ions. RESULTS: The purification coefficient of 29.8-fold with a yield of 9.2% was found. The molecular weight of the purified α-amylase was determined to be 53 kDa by SDS-PAGE, and thermostability was confirmed with 100% activity at 30ºC and 40ºC after 1 h. The purified enzyme was stable over a wide range of pH values, with optimum activity at pH 6.0, 7.0 and 8.0 after 2 h. The study also investigated the effects of the metal ions and detergents on the purified amylase and found that Mg2+ and Ca2+ ions were the activators of the enzyme, while Zn2+, Co2+ and Na+ ions decreased the activity. Furthermore, Hg2+; indicated complete inhibition of amylase activity. The detergents Triton X-100 and Tween 20 increased the α-amylase activity, while sodium dodecyl sulfate inhibited the activity. CONCLUSION: The purified α-amylase obtained from Bacillus sp. is considered to be environmentally friendly, can be processed in a short time, and has a low cost.

Purification and characterization of thermostable α-amylase produced from Bacillus licheniformis So-B3 and its potential in hydrolyzing raw starch.

AIMS: This work was achieved to obtain the optimum culture conditions of the thermostable alpha-amylase produced by thermophilic Bacillus licheniformis SO-B3. Furthermore, the α-amylase was purified and then characterized, and also its kinetic parameters were determined. MATERIALS AND METHODS: A new thermotolerant bacteria called Bacillus licheniformis SO-B3 employed in this work was isolated from a sample of thermal spring mud in Sirnak (Meyremderesi). Several parameters such as the impact of temperature, time, and pH on enzyme production were examined. Thin-Layer Chromatography (TLC) was employed to analyze the end-products of soluble starch hydrolysis, and the utilization of purified α-amylase in the clarification of unripe apple juices was studied. KEY FINDINGS: The highest enzyme production conditions were determined as 35 °C, 36th hour, and pH 7.0. Thermostable α-amylase was purified by 70% ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and dialysis, with a 51-purification fold and 30% yield recovery. The Km and Vmax values for this enzyme were 0.004 mM and 3.07 µmol min-1 at 70 °C, respectively. The α-amylase's molecular weight was found as 74 kDa. In addition, α-amylase showed a good degradation rate for raw starch. SIGNIFICANCE: It was hypothesized that Bacillus licheniformis SO-B3 could be used as an α-amylase source. These findings displayed that purified enzyme could be utilized in fruit juice industries for clarification of apple juice and raw starch hydrolyzing.

A Novel Raw Starch Hydrolyzing Thermostable α-Amylase Produced by Newly Isolated Bacillus mojavensis SO-10: Purification, Characterization and Usage in Starch Industries

ABSTRACT The aim of this study is the production, purification, and characterisation of thermostable raw starch hydrolyzing α-amylase produced by Bacillus mojavensis SO-10. The maximum production conditions of α-amylase were found at 36th hour, 35 °C and pH 7.0. We utilized three steps to purify the thermostable α-amylase and as a result, 34-fold and 18% yield were obtained. The molecular weight of purified α-amylase was determined as 73 kD. The Km and Vmax rates were detected as 0.010 mM and 3.38 µmol min−1, respectively. This purified α-amylase exhibited the highest activity at pH 5.0-6.0 and 70 ºC and showed stability over a wide variety of pH and temperature at 4.0-8.0, and 40-50 ºC, respectively. The thermostable purified α-amylase exhibited stability in the presence of denaturing agents and heavy metal ions. The purified enzyme hydrolyzed the raw starches of corn and wheat grains in the ratio of 36.7% and 39.2% respectively. The end-yields of soluble starch hydrolysis were analyzed by thin-layer chromatography (TLC). In addition, the usage of purified α-amylase in clarification of apple juice and domestic washing detergent industries were evaluated.