Hot Spring Enzyme From Türkiye Survives Boiling Temperatures and Harsh Solvents

Deep beneath the geothermal springs of Türkiye, bacteria have been quietly perfecting molecular machinery that chemists and industrial engineers can only dream of replicating in the laboratory. A research team led by scientists at Atatürk University has now isolated one of these microscopic treasures from four separate hot springs, identified it as Bacillus licheniformis, and extracted from it an alpha-amylase enzyme with a remarkable combination of properties: it works best at 90 degrees Celsius, tolerates alkaline conditions, shrugs off short-term exposure to organic solvents, and can be purified in a single elegant step with almost no loss of activity. The findings, published in the journal 3 Biotech, could point the way toward cheaper and more robust enzymes for the starch, detergent, and biofuel industries, where enzymes routinely face punishing temperatures and chemically hostile environments.

Alpha-amylases are among the most heavily used enzymes on the planet. These catalysts chop long starch molecules into shorter sugars, a deceptively simple reaction that underpins the production of glucose syrups, high-fructose sweeteners, ethanol, laundry detergents, paper coatings, and textiles. The trouble is that many industrial processes run at temperatures and pH values that would denature most proteins within minutes. Starch gelatinization, for example, typically requires heating starch slurries well above the boiling point of water, forcing manufacturers to either cool their process streams before adding enzymes or pay a premium for thermostable variants. Enzymes that naturally thrive under these brutal conditions are therefore worth their weight in gold, and the search for new ones has increasingly turned to the most extreme environments Earth has to offer.

That is precisely where the Turkish team went looking. The researchers collected samples from four geothermal springs, environments where thermophilic bacteria have evolved proteins with unusual structural rigidity, and screened the isolates for their ability to secrete alpha-amylase into the surrounding medium. One isolate, designated SO7, stood out. Through a combination of phenotypic testing, biochemical profiling, enzymatic assays, and sequencing of the 16S rRNA gene, the standard molecular barcode for bacterial identification, the team confirmed that SO7 belonged to Bacillus licheniformis, a species with a long and distinguished history in industrial biotechnology. The identification was methodical rather than dramatic, but it laid the groundwork for everything that followed: a rigorous biochemical dissection of the enzyme the bacterium produces.

Purifying enzymes from crude bacterial cultures is traditionally a laborious, multi-step affair involving ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration, each step bleeding away precious protein. The researchers compared several purification strategies, including starch affinity purification, precipitation with ethanol and acetone, and a technique called three-phase partitioning, or TPP. The comparison produced a striking result. TPP, which uses t-butanol and ammonium sulfate to drive proteins into a concentrated interfacial layer between the aqueous and organic phases, delivered a 71-fold purification with an extraordinary 98.6 percent recovery of enzymatic activity. The aqueous phase after TPP treatment showed an activity of 0.1482 units per milliliter and a specific activity of 592.8 units per milligram, figures that compare favorably with published amylase purifications that required far more handling and suffered far greater losses.

The elegance of TPP matters beyond mere laboratory convenience. Because the method concentrates and purifies the enzyme in a single operation, it dramatically reduces the cost and complexity of scaling up production, a persistent bottleneck in translating laboratory enzyme discoveries into commercial products. For an enzyme intended for industrial use, where kilograms rather than milligrams are needed, a purification scheme that recovers nearly all activity in one step is not just a technical nicety; it is the difference between a publishable curiosity and a viable product candidate. The Turkish team’s results suggest that SO7’s amylase could clear that hurdle more easily than most of its competitors.

Once purified, the enzyme revealed its true character. Sodium dodecyl sulfate polyacrylamide gel electrophoresis pinned its apparent molecular mass at approximately 83.17 kilodaltons, consistent with the size range of bacterial alpha-amylases. More importantly, the enzyme’s activity peaked at pH 7.0 and a scorching 90 degrees Celsius, a temperature at which most ordinary proteins would have long since unfolded into useless tangles. The enzyme also retained substantial activity after being pre-incubated at alkaline pH values and elevated temperatures, indicating that its thermal and pH stability is not merely a transient optimum but a durable property of the protein’s folded structure. This combination of near-neutral pH optimum with alkaline tolerance and extreme heat resistance is exactly the profile that starch liquefaction processes demand.

The enzyme’s resilience extended into chemical territory that typically destroys catalytic proteins. After short-term exposure to selected surfactants, inhibitors, metal ions, and organic solvents, the amylase retained meaningful activity, a property that earned it the label of organic solvent-stable. Solvent tolerance is rare among amylases and highly prized, because many industrial reactions and product formulations involve organic compounds that would strip water from a protein’s surface and collapse its structure. Enzymes that can operate in the presence of solvents open the door to novel biocatalytic processes, including the synthesis of chemicals in non-aqueous media and the processing of solvent-containing waste streams. The researchers were careful to note, however, that this tolerance was assessed only under short-term laboratory conditions, and that performance under real industrial process conditions remains to be validated, an honest caveat that distinguishes careful science from promotional hype.

Kinetic analysis added further depth to the enzyme’s profile. When tested against a panel of starch substrates, rice starch produced the highest relative activity, with a Michaelis constant of 3.24 milligrams per milliliter and a maximum velocity of 204.08 millimoles per milliliter per minute. The Km value indicates a reasonable affinity for the substrate, while the Vmax reflects a brisk catalytic rate under saturating conditions. All measurements were obtained from three independent experiments, giving the numbers a statistical footing that single-run assays often lack. Substrate preference matters in practice: starches from different botanical sources vary in granule structure, amylose content, and susceptibility to enzymatic attack, and an enzyme that performs well on rice starch may behave differently on corn, wheat, or potato starch, the workhorses of the industrial starch sector.

What makes this discovery resonate beyond the enzymology community is the broader story it tells about where biotechnology’s raw materials come from. Extremophiles, organisms that flourish in conditions lethal to most life, have become one of the richest sources of industrially useful enzymes, from DNA polymerases that power the polymerase chain reaction to proteases that clean clothes in hot washes. Every geothermal spring, deep-sea vent, and acidic hot pool sampled by microbiologists yields new candidates, and each one carries the evolutionary fingerprints of proteins stabilized by subtle changes in amino acid composition, salt bridges, and hydrophobic packing. The SO7 amylase is a fresh addition to this catalog, and its combination of thermostability, alkaline tolerance, and solvent resistance suggests that Bacillus licheniformis strains in Türkiye’s hot springs have solved, through billions of years of natural selection, engineering problems that synthetic protein designers still struggle with.

The path from a purified enzyme in a Turkish laboratory to an enzyme working inside a starch plant or detergent factory is long, and the authors themselves flag the limitations of their in vitro study. Scale-up of TPP purification, testing against industrially relevant substrate concentrations, long-term stability trials, and cost analysis all lie ahead. Yet the core achievement stands: a thermostable, solvent-tolerant alpha-amylase purified to near homogeneity with nearly complete activity recovery, characterized with quantitative rigor, and sourced from a bacterium that required nothing more than a trip to a hot spring to find. As industries push toward greener, enzyme-driven processes that replace harsh chemical treatments, discoveries like this one quietly expand the toolkit. The next time a glucose syrup or a biofuel ethanol batch is produced at 90 degrees Celsius in an alkaline reactor, the enzyme doing the work may well trace its ancestry to a steaming pool in the Anatolian countryside.

Subject of Research: Purification and biochemical characterization of a thermostable, organic solvent-stable alpha-amylase from Bacillus licheniformis SO7 isolated from geothermal springs in Türkiye

Article Title: Efficient purification and biochemical characterization of a thermostable and organic solvent-stable α-amylase from Bacillus licheniformis SO7

Article References: Efficient purification and biochemical characterization of a thermostable and organic solvent-stable α-amylase from Bacillus licheniformis SO7. (n.d.). https://doi.org/10.1007/s13205-026-05056-8

Image Credits: AI Generated

DOI: 10.1007/s13205-026-05056-8

Keywords: alpha-amylase, Bacillus licheniformis, thermostable enzyme, three-phase partitioning, enzyme purification, organic solvent tolerance, thermophilic bacteria, geothermal springs, biocatalysis, starch hydrolysis, enzyme kinetics, industrial biotechnology