Production, Purification and Characterization of Thermostable Amylase from Thermophilic Bacteria Isolated from Water and Sediments of Wanzaye Hot Spring, Ethiopia

In the world of industrial manufacturing, enzymes act as nature's own tools, speeding up chemical reactions that would otherwise take far too long or require extreme conditions. Among these, amylases are particularly valuable because they break down starch, a common energy source found in plants, into simpler sugars. While these enzymes are useful, many break down or stop working when the temperature rises, which limits their use in processes that require heat. This is where heat-loving, or thermophilic, bacteria come in. Found in extreme environments like hot springs, these microscopic organisms produce enzymes that remain stable and active even at high temperatures. Scientists have long sought to harness these robust biological catalysts to improve efficiency in industries ranging from food processing to detergent manufacturing, but many regions with geothermal activity remain largely unexplored for these specific resources.

Researchers in Ethiopia turned their attention to the Wanzaye Hot Spring, a natural geothermal site in the northwestern part of the country, to investigate whether its waters and sediments harbored bacteria capable of producing such heat-stable enzymes. The team collected samples from the spring, which sits in a cool, sub-humid climate but emits water at a temperature of 42 degrees Celsius. By heating the samples to 80 degrees Celsius, they filtered out ordinary bacteria and fungi, leaving behind only the tough, spore-forming thermophiles. From these survivors, they isolated twenty distinct bacterial colonies and screened them for their ability to dissolve starch. Eight of these showed strong activity, and four specific strains, named WA7, WA10, WA11, and WA12, were selected for deeper study. All four were identified as belonging to the Bacillus genus, a group of bacteria known for their resilience and ability to form protective spores.

To produce the enzymes, the researchers grew these bacteria using a method called solid-state fermentation, which involves cultivating microbes on solid agricultural waste rather than in liquid. They tested three different materials: wheat bran, rice bran, and banana peel. The results showed that wheat bran was the superior choice, supporting the highest enzyme production. The team determined that the bacteria grew best and produced the most enzyme when kept at a temperature of 55 degrees Celsius with a neutral pH of 7, and that the optimal time to harvest the enzyme was after 72 hours of growth. Once the bacteria had done their work, the researchers extracted the crude enzyme mixture and purified it using a process that involved adding salt to precipitate the proteins and then washing them to remove impurities. This purification step increased the concentration of the active enzyme, making it more effective.

The purified enzymes were then put through a series of tests to understand their capabilities. Analysis revealed that the enzymes were roughly 50 kilodaltons in size, a measure of molecular weight, and that they broke down starch primarily into maltose, a type of sugar. When tested for heat tolerance, the enzymes proved remarkably robust. They reached their peak performance at temperatures between 65 and 70 degrees Celsius, depending on the specific bacterial strain. Even more impressively, after sitting at temperatures ranging from 40 to 80 degrees Celsius for an hour, the enzymes retained more than 76.8 percent of their original activity. They also showed stability across a wide range of acidity levels, functioning well in conditions from slightly acidic to slightly alkaline.

Perhaps the most practical demonstration of their potential involved raw enset starch, a staple food crop in Ethiopia. The researchers mixed the purified enzymes with raw enset powder and found that the enzymes could convert the starch into fermentable sugars with an efficiency exceeding 70 percent for all four strains, with the best performer reaching nearly 91 percent. This suggests that these enzymes could be used to process raw starch directly, a process that is often difficult and energy-intensive. The study concludes that the Wanzaye Hot Spring is a valuable, previously underutilized source of industrially relevant bacteria. By using inexpensive agricultural waste like wheat bran to grow these microbes, the researchers demonstrated a cost-effective way to produce enzymes that can withstand the high temperatures required by modern starch-processing industries, offering a sustainable path forward for local and global manufacturing.