Production, purification and characterization of thermostable α-amylase from soil isolate Bacillus sp. strain B-10

RAVINDRA NATH SINGH, ASHUTOSH BAHUGUNA, PEEYUSH CHAUHAN, VIJAY KUMAR SHARMA, SATINDER KAUR, SANTOSH KUMAR SINGH and AMIR KHAN

ABSTRACT

A bacterial strain B-10 that produces α-amylase was isolated from compost and kitchen waste receiving agricultural soil. Based on microbiological and biochemical tests the isolate B-10 was identified as Bacillus sp. Alpha-amylase produced by this isolate was purified by (NH4)2SO4 precipitation and DEAE cellulose ion-exchange chromatography showing 15.91 and 48.21 fold purification, respectively. SDS-PAGE of the purified enzyme confirmed the purification and monomeric nature of the enzyme. The purified α-amylase showed maximum activity at pH 7 and temperature 50°C. The enzyme was significantly active in the temperature range of 30-60°C for the studied period of 2 h. During the incubation of purified enzyme at pH ranging from 5 to 10 for 24 h the maximum stability was observed at pH 7 followed by pH 8, whereas at extreme pH, the stability was very poor. Km and Vmax were found to be 1.4 mg/mL and 6.2 U/mL, respectively.

Key words: Bacillus, α-amylase, SDS-PAGE, ion exchange chromatography, Michaelis constant,Km

Volume 5, Number 1 (2016) pp. 37-43   [ Full Text (PDF) ]