Utilization of Industrial wastes for the production of amylase from thermophillic Actinomycetes

Abstract

Actinomycetes are a facultative thermophilic group of gram-positive bacteria which produce antibiotics and a range of hydrolytic enzymes including amylases, proteases and lipases. Some Actinomycetes are pathogenic but the majority is saprophytic inhabiting soil, water plants and other habitats, where they play an important role in soil structure and composition. In the present study, bacterial strains from three soil samples obtained from a hot water stream were screened for their potential to produce α -amylase. Out of twenty isolated strains, four were selected due to their amylase producing ability as determined through production of a zone of hydrolysis. These strains were identified by morphology, biochemical tests and PCR amplification. Bacterial strain W1 giving a 3.8 mm zone of hydrolysis was identified to be Actinomycete and N3, U1, and U4 giving 3.6, 3.8 and 3.6 mm zones of hydrolysis, respectively were identified as Bacilli. Growth of Actinomycete and amylase production was optimized on four different nutrient media. Highest growth and amylase activity( 3.882 OD and 132.3 AU, respectively after 72 hours at 60oC) were obtained by using medium-4. Conditions for the maximum production of amylase in submerged culture fermentation were optimized using medium-4. It was found that by using 5% inoculum of 30 hours age, amylase activity of 140.8 AU was obtained. Optimization of agitation speed (150 rpm), temperature (60 oC), pH (7.5), starch (2.5 %) and glucose (3%) increased activity from 140.8 AU to 162.4 AU. Growth and amylase production were then optimized by using agro-industrial waste as sole source of carbon and nitrogen in the modified AGS medium. Nine agro-industrial wastes namely soybean meal, wheat bran, peanut meal, mustard meal, sunflower meal, linseed meal, cottonseed meal, corn meal and molasses were used individually and in combinations to optimize amylase production. Soybean meal and wheat bran were found to be more effective nutrient sources with amylolytic activities of 176.2 AU and 171.6 AU respectively. Soybean meal in combination with molasses at 2:1 ratio were found to bethe best carbon and nitrogen sources giving amylolytic activity of 230.6 AU. In solid state fermentation, maximum amylolytic activity( 186.2 AU) was obtained by using wheat bran and molasses in 2:1 ratio at 55oC after 72 hours of incubation. The enzyme was purified 24-fold by acetone precipitation and gel filtration. Single peak during gel filtration indicated presence of only one type of alpha-amylase. Crude and partially purified enzyme find vital application in desizing of cotton cloth. Desizing of cotton pieces 15x15 cm by acid hydrolysis (0.1 M HCl) resulted in 0.7725g removal of starch. This value served as standard for 100% starch removal. Crude enzyme gave 98.39% removal after 90 minutes of soaking at pH 7.0. However, 98.92 % removal was achieved with partially purified enzyme after 60 minutes of soaking. Optimization of temperature and pH revealed that 100 % desizing was obtained at pH 6.0 when incubated at 60oC for 60 minutes. Studies proved to be successful in utilizing agro-industrial waste for producing thermostable alpha-amylase from Thermoactenomyces sacchari and its application as a desizer. The process is recommended for large production of thermostable alpha-amylase in the future.