Adenosine triphosphate (ATP) acts as a crucial energy currency in vivo, and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0–9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively. Graphical Abstract

A rapid in vitro enzymatic biosynthesis system has been developed as a biological manufacturing platform with potential industrial uses. Cytidine 5′-monophosphate (5′-CMP) is a key intermediate in the preparation of several nucleotide derivatives and is widely used in food and pharmaceutical industries. In this study, a highly efficient biosynthesis system was constructed for manufacturing 5′-CMP in vitro. Cytidine kinase (CK) was used for the biotransformation of cytidine to 5′-CMP, while polyphosphate kinase (PPK) was coupled for adenosine triphosphate regeneration. Both CK and PPK were selected from extremophiles, possessing great potential for biocatalytic synthesis. The effects of temperature, substrate concentration, and enzyme ratios were investigated to enhance the titer and yield of 5′-CMP. After optimization, 96 mM 5′-CMP was produced within 6 h, and the yield reached nearly 100%. This work highlights the ease of 5′-CMP production by an in vitro biomanufacturing platform and provides a green and efficient approach for the industrial synthesis of 5′-CMP.

A series of cross-linked polyurethane(PU) elastomer samples with various crosslink density were synthesized from polyether diol(PPG2000), 4,4’-diphenylmethane diisocyanate(MDI), 1,4-butanediol(BDO) , trimethylolpropane (TMP) and glycerin. The cross-linking density of the PU elastomers was calculated by Flory–Rehner equation. The degree of hydrogen bonding, the microstructure and the morphologies of these PU materials were characterized by means of FT-IR, DSC and DMA. The experimental results showed that the PU elastomers containing a small amount of crosslink agent ( TMP or glycerin ) may make tanδ to a very low value above the ambient temperature. The PU elastomer samples using glycerin as curing agent can make tanδ to a low value in a lower temperature compared with the ones using TMP as curing agent.

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