Abstract
Pyrimidine-nucleoside phosphorylases (Py-NPases) have a significant potential to contribute to the economic and ecological production of modified nucleosides. These can be produced via pentose-1-phosphates, an interesting but mostly labile and expensive precursor. Thus far, no dynamic model exists for the production process of pentose-1-phosphates, which involves the equilibrium state of the Py-NPase catalyzed reversible reaction. Previously developed enzymological models are based on the understanding of the structural principles of the enzyme and focus on the description of initial rates only. The model generation is further complicated, as Py-NPases accept two substrates which they convert to two products. To create a well-balanced model from accurate experimental data, we utilized an improved high-throughput spectroscopic assay to monitor reactions over the whole time course until equilibrium was reached. We examined the conversion of deoxythymidine and phosphate to deoxyribose-1-phosphate and thymine by a thermophilic Py-NPase from Geobacillus thermoglucosidasius. The developed process model described the reactant concentrations in excellent agreement with the experimental data. Our model is built from ordinary differential equations and structured in such a way that integration with other models is possible in the future. These could be the kinetics of other enzymes for enzymatic cascade reactions or reactor descriptions to generate integrated process models.
Highlights
Pyrimidine-nucleoside phosphorylases (Py-NPases) are highly versatile enzymes used for the production of pharmaceutically relevant nucleoside derivatives and pentose-1-phosphates
We present experimental data deduced from the reaction monitoring of small-scale Py-NPase reactions via a UV/Vis spectroscopy-based assay
In the tested range of enzyme and substrate concentrations, we found an excellent agreement between experimental data and our model with this parameter set (Figure 3)
Summary
Pyrimidine-nucleoside phosphorylases (Py-NPases) are highly versatile enzymes used for the production of pharmaceutically relevant nucleoside derivatives and pentose-1-phosphates. Nucleoside phosphorylases catalyze, in the presence of phosphate, the reversible conversion of a nucleoside to the corresponding pentose-1-phosphate and nucleobase (Figure 1). Due to the low yields of modified nucleosides or pentose-1-phosphates via conventional synthetic chemistry, nucleoside phosphorylases have become attractive tools in their biocatalytic preparation [1,2,3]. Thermophilic Py-NPases have attracted increased interest, as they combine several favorable properties, Processes 2019, 7, 380; doi:10.3390/pr7060380 www.mdpi.com/journal/processes. Processes 2019, 7, 380 such as long shelf life due to their thermal stability, an excellent tolerance towards harsh reaction conditions, high turnover rates, and a broad substrate spectrum [4,5]. Processes 2019, 7, x FOR PEER REVIEW (a) (b) Figure 1.
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