Purification, characterization and potential detergent industry application of a thermostable α-amylase from Bacillus licheniformis RA31

alpha-Amylase is an endoamylase catalysing the degradation of starch into maltose, maltotriose and glucose. The enzyme isolated from microbial sources possess unique properties of thamostability thus making it a useful tool in the detergent industry. Here, we explored a strain of thermophilic bacteria Bacillus licheniformis for its potential application in detergent industry. The B. licheniformis RA31 was isolated from soil samples of hot spring in Rampur district of Himachal Pradesh, India and grown on optimized media to produce thermostable alpha-amylase. The enzyme was ethanol precipitated, purified (12.93 fold, 55.52% yield and 621.93 U/mg specific activity) to homogeneity with a single band on SDS-PAGE (66 kDa) and native-PAGE (68 kDa). Purified enzyme displayed best activity in pH 8 buffer and similar to 80% activity was retained in pH 7 and 10. It showed temperature optima at 70 degrees C. Its activity was decreased at 70 degrees C (70% after 4 h), 80 degrees C (65% after 4 h) and 90 degrees C (50% after 1 h). The enzyme was stimulated (126%; 5 mM) by barium chloride. It was relatively stable in the presence of commercial detergents (109-125%), SDS (84%), Tween 20 (88%), EDTA (72%) and beta-ME (70% at 10 mM). K-m and V-max for the enzymatic hydrolysis of starch were 0.339 mg/mL and 1.450 mg/min, respectively. The enzyme revealed the highest specificity towards wheat starch granule (140% after 1 h) and SEM analysis displayed its biodegradation (2-10 h). Improved cleaning efficiency of potato curry stained fine cotton clothes were observed with enzyme assisted detergent advance treatment (0.02% w/v). The enzyme showed potential applications in detergent industry.