Japan-based scientists have developed an enzyme that can convert ferulic acid from plant waste into vanillin, a flavor compound widely used in food and beverage products. The sweet smell of vanilla extract is imparted by the chemical compound vanillin, which is found in the seed pods of vanilla plants. The enzyme VpVAN synthesizes vanillin by converting ferulic acid.

However, laboratory biosynthesis of vanillin from plant-derived VpVAN yields only very small quantities of vanillin and is considered commercially impractical. In addition, cheaper chemically derived vanilla essences often do not match the flavor of natural vanilla extract.

Enzyme-made sustainable vanillin
In addition, the vanilla supply chain is well known for price fluctuations and volatile market dynamics, partly due to vanilla plants producing such small yields, leading to dwindling supply and price surges. The rapid onset of climate change and extreme weather events also affect cultivation.

These factors led to the study by a research team at Tokyo University of Science (TUS), recently published in Applied and Environmental Microbiology.

They adopted a new method that uses abundant agricultural waste, potentially reducing environmental impact and supply chain costs. It is also hailed as a “breakthrough” by TUS.

A “revolution” for vanilla production
Professor Toshiki Furuya from TUS tells Food Ingredients First about the development that addresses the vanilla sector’s challenges.

“Vanillin is available from the beans of the vanilla orchid through extraction. However, the yield of vanillin is very low because it accumulates at low levels in the plant. Biotechnological production of vanillin using enzymes has attracted much attention as an alternative to extraction from vanilla beans,” he explains.

“Ferulic acid is a practical starting material for the biotechnological production of vanillin because a large amount of this compound can be recovered from agroindustrial wastes, including wheat and rice bran.

“Several multiple microbial-enzyme systems have been reported in biocatalytic processes to produce vanillin from ferulic acid. However, systems that use multiple enzymes are generally complex and expensive because multiple enzymes need to be prepared, and reaction conditions must be adjusted for each enzyme,” Furuya continues.

Cost-effective method
To date, however, no microbial enzyme that synthesizes vanillin from ferulic acid in one step has been reported.

Furuya notes that if vanillin synthesis from ferulic acid could be accomplished with a single enzyme, it would further streamline vanillin production.

“In this study, we succeeded in developing a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner. This means that vanillin can be produced simply by mixing the enzyme and ferulic acid at room temperature. Our approach using the enzyme and agroindustrial wastes offers a sustainable and cost-effective method for vanillin production.”

The researchers used genetic engineering approaches to modify the molecular structure of an enzyme, “Ado,” originally an oxidase enzyme that adds an oxygen atom to the substrate, isoeugenol.

In its native state, Ado does not have the ability to convert ferulic acid and vanillin. However, Furuya used a structural modeling analysis to predict amino acid changes in Ado that allowed it to interact with ferulic acid.

They trialed replacing phenylalanine and valine amino acid residues at specific positions in the structure of Ado with various other amino acids. Their experiments then examined the ferulic acid conversion ability of the various engineered mutant proteins.

Eventually, they discovered a mutant protein in which only three specific phenylalanine and valine residues were replaced with tyrosine and arginine. This protein stably reacted with ferulic acid and exhibited high conversion activity.

Notably, unlike other oxidases, the engineered enzyme did not require any cofactors for conversion and produced vanillin on a gram scale per liter of reaction solution, with a higher catalytic efficiency and affinity than that of the wild-type enzyme.

The reaction only required mixing of the enzyme, ferulic acid, and air (molecular oxygen) at room temperature, making it a simple, sustainable and economically scalable process.

Stabilizing supply
As the yield of vanillin obtained from the beans of the vanilla orchid is very low and dependent on the weather, its supply is unstable.

“Our approach is expected to provide a stable supply of vanillin because the starting material, ferulic acid, is easily obtained from agroindustrial wastes, and this method is not affected by the weather.”

“Currently, vanillin on the market is mainly produced either by extraction from plants or chemical synthesis. We consider that our approach can provide a new option, “enzyme-made sustainable vanillin,” Furuya concludes.