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

Abstract

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 Şırnak (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 K_m and V_max 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.

Keywords: Apple juice; Bacillus licheniformis; Chromatography; Raw starch; α-Amylase.