RESUMO
?-Amylase is an endoamylase catalysing the degradation of starch into maltose, maltotriose and glucose. The enzyme isolated from microbial sources possess unique properties of thermostability 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 ?-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 ?80% activity was retained in pH 7 and 10. It showed temperature optima at 70°C. Its activity was decreased at 70°C (70% after 4 h), 80°C (65% after 4 h) and 90°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 ?-ME (70% at 10 mM). Km and Vmax 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.
RESUMO
Among different types of microbial enzymes, amylases are the most widely used in industries as they are produced in large quantity and in an economic way as compared to plants and animals. Moreover, thermostable amylase has significance as compared to the amylase from mesophiles. Therefore, hot water springs are explored to dig into its bacterial diversity. In the current study, we tried to isolate amylase producing bacteria from the soil and water samples collected from the hot water spring in Rampur, Himachal Pradesh, India. The samples were serially diluted before plating on the Luria Bertani agar plates. A total of 42 bacterial morphotypes were isolated and were screened for amylolytic activity by starch agar plate method. Among the 42 bacterial isolates 25 showed amylolytic activity. Production of amylase was carried out at different temperatures and pH to optimize the temperature and pH conditions for each isolate. All the 25 isolates were characterized based on morphology, biochemical tests and molecular analysis. Sequencing of 16S rRNA gene for the 25 isolates followed by BLAST search revealed a majority of them (19) identified as Bacillus licheniformis. Other isolates were identified as B. subtilis, B. safensis, B. halodurans, B. stratosphericus, Caldimonas hydrothermale and Exiguobacterium mexicanum. An attempt was made to amplify amyN gene which codes for α-amylase but successful amplification was achieved only from Bacillus licheniformis alone.