Natural vanillin extracted from plants rapidly using a Bioengineered enzyme

Natural vanilla extract is becoming harder to get and costs more money because of climate limitations on growing the plant and the low yields that each plant produces.

One of the most popular flavouring ingredients in food items and cosmetics is vanilla extract. The chemical ingredient called “vanillin,” which is present in the seed pods of vanilla plants that belong to the orchid family, gives this traditional flavour its pleasant and sweet aroma. Plants produce vanillin by the enzyme VpVAN converting ferulic acid. Nevertheless, vanillin produced by laboratory biosynthesis from plant-derived VpVAN is not economically viable due to the extremely low yield of vanillin. Moreover, real vanilla extract is still in high demand even if chemically produced vanilla essences are less expensive and do not have the same flavour. Natural vanilla extract is becoming harder to get and costs more money because of climate limitations on growing the plant and the low yields that each plant produces.

In response to these difficulties, Professor Toshiki Furuya of the Tokyo University of Science’s Department of Applied Biological Science, Faculty of Science and Technology, and his graduate students Shizuka Fujimaki and Satsuki Sakamoto created an enzyme that converts ferulic acid derived from plants into vanillin.

Ferulic acid, the raw material, is a compound that can be obtained in abundance from agricultural waste such as rice bran and wheat bran. Vanillin is generated simply by mixing ferulic acid with the developed enzyme at room temperature. So, the established technology can provide a simple and environmentally friendly method for producing flavor compounds.

Prof. Furuya

The detailed study report of the project is published in the journal Applied and Environmental Microbiology.

The researchers altered the molecular structure of the enzyme “Ado” by genetic engineering techniques. Ado was first identified as an oxidase enzyme that increases the amount of oxygen in the substrate, isoeugenol. It is incapable of converting ferulic acid into vanillin in its natural state. The researchers were able to forecast changes in Ado’s amino acid composition that would allow it to interact with ferulic acid through the use of structural modelling analysis. In keeping with this, scientists carried out a number of tests in which they substituted other amino acids for the phenylalanine and valine amino acid residues at particular locations in the Ado structure. They next investigated the different designed mutant proteins’ capacity to convert ferulic acid.

After several trials and errors, they discovered that a mutant protein with high conversion activity and a stable reaction with ferulic acid was one in which just three particular phenylalanine and valine residues were swapped out for tyrosine and arginine. Interestingly, the modified enzyme generated vanillin on a Gramme scale per litre of reaction solution, with a better catalytic efficiency and affinity than that of the wild-type enzyme. It also did not require any cofactors for conversion, in contrast to other oxidases. The reaction was straightforward, environmentally friendly, and economically scalable because it just needed the enzyme, ferulic acid, and air (molecular oxygen) to be mixed at ambient temperature.

Additionally, the molecularly developed enzyme demonstrated conversion ability towards chemicals derived from the breakdown of lignin, a typical agricultural waste product, such as p-coumaric acid and sinapic acid.

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At this point, no enzymes produced by microorganisms or plants have demonstrated the capacity to industrially convert ferulic acid to vanillin. As a result, the enzyme created in this work has a great deal of promise to make it possible to produce natural vanillin in a way that is both profitable and sustainable.

Harnessing the potential of microorganisms and enzymes to derive valuable compounds under mild conditions from renewable plant-based resources, offers a sustainable approach to minimizing environmental footprint. Presently, in collaboration with a company, our research endeavors focus on achieving the real-world implementation of vanillin production through the utilization of the newly developed enzyme.

Prof. Furuya

Source: Tokyo University of Science Media Relations.

Journal Reference: Fujimaki S, Sakamoto S, Shimada S, Kino K, Furuya T.0. Engineering a coenzyme-independent dioxygenase for one-step production of vanillin from ferulic acid. Appl Environ Microbiol0:e00233-24.

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