Potential of Endophytic Fungi from Orobanche spp. as Novel Sources of L-Asparaginase, an Enzyme with Anticancer Applications

In the hidden world inside healthy plants, tiny fungi live as quiet neighbors, sharing space with their hosts without causing harm. These are endophytes, a diverse community of microorganisms that have evolved to produce a vast array of chemical tools to help them survive and thrive. Among the many substances these fungi create are enzymes, which are biological machines that speed up specific chemical reactions. One such enzyme, known as L-asparaginase, has become a critical tool in modern medicine. It works by breaking down a specific building block of proteins called L-asparagine. While most cells in the human body can make their own supply of this building block, certain cancer cells, particularly those involved in a type of blood cancer called acute lymphoblastic leukemia, cannot. They rely entirely on stealing it from the blood. By introducing the enzyme into a patient's system, doctors can strip away this essential nutrient, causing the cancer cells to starve and die while leaving healthy cells largely unaffected. Currently, the medical industry produces this life-saving enzyme primarily from bacteria, but these bacterial versions can sometimes trigger severe allergic reactions or other side effects in patients, prompting scientists to search for gentler, more compatible sources.

A team of researchers at the University of Zanjan in Iran turned their attention to an unusual and often overlooked host for these microscopic neighbors: the broomrape plant. Unlike most plants that grow from the soil, broomrape is a holoparasite, meaning it has no leaves or roots of its own and survives entirely by attaching to the roots of other crops and stealing their nutrients. Because of this unique and stressful lifestyle, the fungi living inside broomrape might have developed special abilities to cope with their environment. The researchers collected samples of healthy broomrape plants from agricultural fields in northwestern Iran and carefully cleaned them to remove any surface dirt or bacteria. They then cut the plant stems into small pieces and placed them on a nutrient-rich jelly to see if any fungi would grow out from the inside. From 300 plant samples, they successfully isolated dozens of different fungal strains, eventually grouping them into 34 distinct types based on how they looked under a microscope.

To find out which of these fungi could produce the cancer-fighting enzyme, the scientists first used a simple visual test. They grew the fungi on a special plate containing L-asparagine and a pH indicator dye. If a fungus produced the enzyme, it would break down the asparagine and release ammonia, which would turn the surrounding area from yellow to pink. About 20 of the 34 fungal types created these pink halos, suggesting they were capable of making the enzyme. However, the researchers knew that a large pink circle on a plate does not always mean a high amount of enzyme is being produced. To get the true measure, they moved the most promising candidates into liquid cultures and used a chemical method to precisely count how much ammonia was released. The results showed a wide range of activity. While some fungi produced very little, four specific strains stood out as the most productive. Among them, a fungus identified as Alternaria longipes, specifically a strain labeled SH25, produced the highest amount of enzyme, reaching 5.79 units per milliliter. This was significantly more than the other top performers, which included species like Macrophomina phaseolina and Aspergillus flavus.

The team then focused on the star performer, the Alternaria longipes SH25, to see if they could make it produce even more enzyme by changing its environment. They tested how the amount of food available and the acidity of the liquid affected production. They found that the fungus produced the most enzyme when the liquid was slightly acidic and contained a specific concentration of L-asparagine. Under these optimized conditions, the enzyme output rose to 5.95 units per milliliter. The researchers also checked how well the enzyme held up when exposed to heat, a crucial factor for industrial use. They found that the enzyme remained very stable at moderate temperatures, retaining almost all of its activity after being held at 35 degrees Celsius for half an hour, and still keeping about two-thirds of its power after two hours. This suggests the enzyme is robust enough to be handled in a manufacturing setting without breaking down immediately.

The study concludes that the parasitic broomrape plant is a rich, yet previously unexplored, source of fungi capable of producing this vital enzyme. The discovery of the high-performing Alternaria longipes strain offers a potential new alternative to the bacterial sources currently used in medicine. While the bacterial versions can cause immune reactions in some patients, fungal enzymes are often considered to be more compatible with the human body, though further testing is needed to confirm this advantage. The researchers noted that the size of the pink halo on the initial test plates was not a reliable predictor of how much enzyme the fungus would actually produce in a liquid culture, highlighting the importance of precise chemical measurements over visual guesses. By isolating and identifying these fungi using genetic sequencing, the team confirmed their identities and established a clear link between this specific fungal species and its ability to produce the enzyme. This work opens a new door for biotechnology, suggesting that even the most unusual plants in our fields may harbor microscopic allies capable of helping to fight disease.