Statistical Approach to Enhance α-Amylase Production from Bacillus licheniformis and Purification of the Enzyme

Abstract

The most widely used thermostable enzymes are the amylases in the starch industry. These are among the most important enzymes and are of great significance in present day biotechnology. The main objective of the present study was to enhance α-amylase production from Bacillus licheniformis using Response Surface Methodology (RSM) and the purification of the enzyme to homogeneity. Bacterial culture producing α-amylase and isolated from hot spring (Himachal Pradesh) was identified as Bacillus licheniformis using 16S rDNA gene sequencing (NCBI Accession No.: KR340466). Medium components and pysical culture parameters viz. pH, temperature, inoculum size, peptone concentration and starch concentration were optimized using RSM. Among these five factors, three factors (starch concentration, peptone concentration and inoculum size) had positive effect on amylase production. A 4.09-fold increase in production of α-amylase from B. licheniformis was achieved using RSM as compared to One Factor At a Time. The enzyme was purified by using Diethylaminoethyl Cellulose column chromatography and subsequently by Sephadex G-75 gel filtration chromatography. A purification fold of 23.39 and a yield of 12.12% was observed. A single band of 33 kDa was obtained using Sodium Dodecyl Sulphate (SDS) and native-Poly Acrylamide Gel Electrophoresis (PAGE) which indicated that the enzyme was purified to homogeneity and was a monomer. The enzyme showed stability at 50 and 65°C temperatures and at alkaline pH. The stability of purified enzyme at high temperatures and alkaline pH suggested its wide application in textile, detergent and paper industries. Keywords: Amylase; Bacillus licheniformis; RSM; SDS-PAGE; alkaliphilic; thermostable enzymes