What is it about?
The enzymatic synthesis of nucleotides offers several advantages over traditional multistep chemical methods, such as stereoselectivity, regioselectivity, enantioselectivity, simple downstream processing, and the use of mild reaction conditions. However, in order to scale up these bioprocesses, several drawbacks, such as the low enzyme stability and recycling, must be considered. Enzyme immobilization may overcome these cost-related problems by enhancing protein stability and facilitating the separation of products. In this regard, tetrameric hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRT) from Thermus thermophilus HB8 was covalently immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles (MTtHGXPRT). In this context, two different strategies were followed: (a) an enzyme immobilization through its N-terminus residues at pH 8.5 (derivatives MTtHGXPRT1-3); and (b) a multipoint covalent immobilization through the surface lysine residues at pH 10 (derivatives MTtHGXPRT4-5). The immobilized derivatives of MTtHGXPRT3 (activity 1581 international units per gram of support, IU/g; retained activity 29%) and MTtHGXPRT5 (activity 1108 IU/g; retained activity 23%) displayed the best wet biocatalyst activity, and retained activity values in the enzymatic synthesis of inosine-5-monophosphate (IMP). In addition, the dependence of the activities and stabilities of both derivatives on pH and temperature was tested, as well as their reusability potential. Taking these results into account, MTtHGXPRT3 was chosen as the best biocatalyst (negligible loss of activity at 60 Cduring24h;reusable up to seven cycles). Finally, as proof of concept, the enzymatic production of dietary nucleotides from high concentrations of low soluble bases was achieved.
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Why is it important?
The enzymatic synthesis of nucleotides is important because it offers several key advantages over traditional chemical synthesis methods: it provides stereoselectivity, regioselectivity, and enantioselectivity, uses mild reaction conditions, and allows simpler downstream processing. These features make enzymatic processes more efficient, environmentally friendly, and suitable for producing bioactive nucleotides and their analogs important in biology and medicine. However, scaling up enzymatic processes faces challenges, notably enzyme stability and recycling. Immobilizing enzymes on supports can enhance their stability, facilitate product separation, and enable enzyme reuse, which lowers costs and improves process sustainability. Immobilized enzymes typically show better operational stability, can be reused for multiple reaction cycles, and reduce enzyme loss during processing. This makes enzymatic synthesis more economically viable and supports large-scale production of valuable nucleotides, such as inosine-5-monophosphate (IMP), as demonstrated with the immobilized HGXPRT enzyme from Thermus thermophilus in the research described. In summary, enzymatic synthesis combined with enzyme immobilization is important because it enables efficient, selective, and sustainable production of nucleotides with enhanced enzyme stability and reusability, thereby overcoming major limitations of free enzyme usage in industrial bioprocesses.
Perspectives
As the author of this article, my personal perspective is that this research clearly and practically demonstrates the value of the covalent immobilization of the tetrameric enzyme HGXPRT from Thermus thermophilus HB8. I immobilize the enzyme onto glutaraldehyde-activated magnetic microparticles, which significantly improves its stability and allows for its reuse without significant loss of activity. This is essential to making the enzymatic synthesis of nucleotides, such as inosine-5-monophosphate (IMP), a viable and sustainable industrial process. I highlight that these results offer effective solutions to common problems in large-scale enzymatic production, such as low stability and difficulty recovering the enzyme. Additionally, the immobilized biocatalyst's ability to perform well under different pH and temperature conditions and to be reused over multiple cycles is especially relevant for practical applications. Finally, I am pleased to see that synthesizing nucleotides from poorly soluble bases is possible, which broadens the options for industrial production and commercial applications in pharmaceutical and food sectors. In short, I consider this publication a valuable advance in the field of industrial biocatalysis with an innovative and applicable approach.
Octavio Corral Pazos de Provens
Universidad Internacional de La Rioja
Read the Original
This page is a summary of: One-Pot, One-Step Production of Dietary Nucleotides by Magnetic Biocatalysts, Catalysts, April 2018, MDPI AG,
DOI: 10.3390/catal8050184.
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