Unlocking natural TMP with sustainable enzyme technology
Dr Valentina Jurkaš and Priv.-Doz. Dr Margit Winkler from ACIB present sustainable enzymatic synthesis of tetramethylpyrazine for flavour and pharma *
Tetramethylpyrazine( TMP) has long been valued across industries( Figure 1). In the food sector, it contributes nutty, roasted, and toasted flavour notes characteristic of heatprocessed foods. 1, 2
In traditional Chinese medicine, where it occurs naturally in Ligusticum wallichii, TMP is valued for its cardioand cerebrovascular benefits. Beyond this, TMP exhibits anticancer activity and serves as a building block for pharmaceutically active compounds. 1- 3
Demand for natural TMP is far beyond what can be extracted sustainably from natural sources. Current biotechnological methods focus on boosting microbial acetoin production, which can then condense with ammonia under high-temperature conditions. 1, 2, 4
While effective, these processes face limitations in scalability, control over by-products and reliance on harsh conditions, all of which restrict their suitability for sustainable manufacturing.
A mild, enzymatic solution
Our research explores a fully enzymatic cascade, operating under mild, aqueous conditions. This cascade involves three distinct biocatalytic steps followed by spontaneous conversion( Figure 2): 1. Carboligase( CL) catalyses decarboxylation and C – C bond formation, producing a short-chain hydroxyketone
2. Transaminase( TA) introduces the amino group, converting the hydroxyketone into a vicinal amino alcohol
3. Alcohol dehydrogenase( ADH) oxidises the vicinal amino alcohol to a reactive aminoketone intermediate
Figure 1- Applications of TMP
4. Spontaneous condensation – under mild aqueous conditions, the aminoketone dimerises to form TMP
In initial studies, each enzymatic step was first evaluated individually, using a panel of candidate enzymes to identify the most effective biocatalyst for each transformation.
CLs are remarkable enzymes capable of decarboxylating an α-keto acid, in this case pyruvic acid, and simultaneously coupling the resulting carbanion to an acceptor aldehyde, forming a new C – C bond.
This reaction not only establishes the carbon framework for TMP but also allows us to start from a simple, naturally derived substrate. The use of pyruvic acid provides a renewable, low-cost entry point, consistent with the sustainability principles guiding this project.
To identify a robust biocatalyst for this key transformation, we explored a diverse range of known CLs and also uncovered several new enzymes with promising activity. 5- 7 The selected CL achieved up to 77 % yield, providing a strong foundation for the subsequent steps.
The TA reaction also proceeded smoothly, reaching 80 % yield, while the final ADH oxidation presented a greater challenge. As this type of oxidation is rarely reported in the literature, identifying an active enzyme required extensive screening.
Using a racemic amino alcohol standard, we nevertheless achieved up to 46 % TMP yield( Figure 3). A dedicated protein engineering campaign is envisioned in order to alleviate this bottleneck and improve the performance of the ADH step.
Enzymatic cascade development
Having identified suitable enzymes for each reaction individually, the next challenge was to integrate them into a single, streamlined process. Developing a compatible multi-enzyme cascade requires careful balancing of reaction conditions to ensure that all catalysts can function efficiently in a shared environment.
Enzymes, by nature, operate under mild aqueous conditions, often at moderate temperatures and nearneutral pH. However, aligning the optimal parameters of several enzymes within one process can be complex.
While the CL and TA both performed well at 28 ° C and neutral pH, running both simultaneously led to unintended transamination and depletion of the starting material. In addition, the subsequent alcohol oxidation required a basic pH and acetone as a co-substrate to enable nicotinamide cofactor recycling.
44 SPECIALITY CHEMICALS MAGAZINE ESTABLISHED 1981