Production of Amylase from Thermophilic Bacteria from Harmukale Natural Hot Spring in Somali Region, Ethiopia

Abstract

Enzymes are biological catalysts, which initiate and speed up thousands of biochemical reactions in living cells. Among industrially important enzymes, amylases, protease and cellulases are getting more attention and importance because these enzymes have wide applications in many fields. For instance, amylases are used in various industries such as food, detergent, paper, textile, beverage, pharmaceutical and fine-chemical industries. Thermophiles are a potent source of thermos enzymes, which show at most stability and activity under conditions of high temperature. The aim of this research was to produce amylase from thermophilic bacteria from hot spring of Harmukale (Dembel), Somali region, Ethiopia. Sediment l samples were collected randomly in triplicate from Harmukale naturally hot springs, Ethiopia. From these sediment samples, thermostable amylase-producing bacteria were isolated by the serial dilution method. Pure colonies produced by streak plate was transferred into freshly prepared starch agar plates and incubated at 50°C for 48 hrs. The plates were flooded with 1% iodine solution. Clear zone formation around the colonies, which suggests starch hydrolysis, was observed and the diameter of the clear zone was measured. A total of 30 different colonies were identified based on colony shape. Ten (33%) of these isolates were shown to provide a zone of clearing surrounding their colonies when the iodine solution was applied to them. The three isolates, I1, I2, and I3, were chosen for further investigation because, compared to the other screened isolates, they produced the largest ratio of halo diameter. The isolates I1, I2, and I3 were classified as Gram-positive, rod-shaped arranged in chains, and spore-forming bacterial species that may belong to the genus Bacillus. Also, these isolates have been identified as motile. Various biochemical tests were performed, the three isolate (I1, I2, and I3) displayed positive result for methyl red, citrate utilization, catalase, urease and starch hydrolysis tests. The crude enzymes were produced from three potential thermophilic bacterial isolates under submerged condition. Culture filtrates were separated by centrifugation at 10000 rpm for 10 min and the supernatants containing cell free extract were used as crude enzyme source for testing the activity on white clothes stained by chocolate. Then Clothes with chocolate stains were employed for the activity test of crude thermostable amylase made from possible isolates, and the amylase was used to get rid of stubborn stains. Even though stain removal took different amounts of time, isolate I1 and I2 removed chocolate stains wit in 120 min whereas isolate I2 removed stain for 240 min. which mean I1 and I2 removed the stain with short time than I3.