“…This is in good accordance with previous observations where protein leach from the matrix was observed when non-covalent immobilization was applied [30]. Due to the limitations of non-covalent binding, post-immobilization by cross-linking and covalent binding were developed to avoid desorption of enzyme under more harsh conditions [19,[31][32][33]. As an example, Serra and colleagues compared different strategies to cross-link with oxidized dextran [33].…”
Section: Discussionsupporting
confidence: 86%
“…After immobilization to Ni-NTA superflow, the substrate spectrum of the immobilized HsdCK was not affected; however, the conversion percentages and the enzyme-specific activity was decreased in comparison to the soluble enzyme. Although, ionic adsorption to charged supports is described to be a mild way of enzyme immobilization [19], our results showed a decreased substrate conversion percentages using low substrate concentrations. This might be explained by mass-transfer limitations of the substrate to contact the enzyme [22] especially as the applied Ni-NTA sepharose is a gel-like matrix.…”
Section: Discussioncontrasting
confidence: 52%
“…Furthermore, dCK activity is essential for the activation of a wide variety of important therapeutic prodrugs, such as the antiviral agent lamivudine (3TC), for the treatment of HIV infection [16], or antineoplastic agents like cladribine [17] (Scheme 1). Both whole immobilized cells and purified enzymes have been used for the synthesis of NMP analogues [18,19]. Immobilization techniques help to stabilize biocatalysts under harsh conditions (e.g., high temperature, extreme pH, solvents) [19].…”
Section: Introductionmentioning
confidence: 99%
“…Both whole immobilized cells and purified enzymes have been used for the synthesis of NMP analogues [18,19]. Immobilization techniques help to stabilize biocatalysts under harsh conditions (e.g., high temperature, extreme pH, solvents) [19]. As the immobilized biocatalysts can be used repeatedly, the processes get more cost-effective, which is an important consideration for industrial applications.…”
Section: Introductionmentioning
confidence: 99%
“…Nickel-nitrilotriacetic acid (Ni-NTA) superflow was applied as the support, since the applied enzymes bear an N-terminal His-tag and it is described as a gentle way of immobilization. Both whole immobilized cells and purified enzymes have been used for the synthesis of NMP analogues [18,19]. Immobilization techniques help to stabilize biocatalysts under harsh conditions (e.g., high temperature, extreme pH, solvents) [19].…”
Natural ribonucleoside-5’-monophosphates are building blocks for nucleic acids which are used for a number of purposes, including food additives. Their analogues, additionally, are used in pharmaceutical applications. Fludarabine-5´-monophosphate, for example, is effective in treating hematological malignancies. To date, ribonucleoside-5’-monophosphates are mainly produced by chemical synthesis, but the inherent drawbacks of this approach have led to the development of enzymatic synthesis routes. In this study, we evaluated the potential of human deoxycytidine kinase (HsdCK) as suitable biocatalyst for the synthesis of natural and modified ribonucleoside-5’-monophosphates from their corresponding nucleosides. Human dCK was heterologously expressed in E. coli and immobilized onto Nickel-nitrilotriacetic acid (Ni-NTA) superflow. A screening of the substrate spectrum of soluble and immobilized biocatalyst revealed that HsdCK accepts a wide range of natural and modified nucleosides, except for thymidine and uridine derivatives. Upon optimization of the reaction conditions, HsdCK was used for the synthesis of fludarabine-5´-monophosphate using increasing substrate concentrations. While the soluble biocatalyst revealed highest product formation with the lowest substrate concentration of 0.3 mM, the product yield increased with increasing substrate concentrations in the presence of the immobilized HsdCK. Hence, the application of immobilized HsdCK is advantageous upon using high substrate concentration which is relevant in industrial applications.
“…This is in good accordance with previous observations where protein leach from the matrix was observed when non-covalent immobilization was applied [30]. Due to the limitations of non-covalent binding, post-immobilization by cross-linking and covalent binding were developed to avoid desorption of enzyme under more harsh conditions [19,[31][32][33]. As an example, Serra and colleagues compared different strategies to cross-link with oxidized dextran [33].…”
Section: Discussionsupporting
confidence: 86%
“…After immobilization to Ni-NTA superflow, the substrate spectrum of the immobilized HsdCK was not affected; however, the conversion percentages and the enzyme-specific activity was decreased in comparison to the soluble enzyme. Although, ionic adsorption to charged supports is described to be a mild way of enzyme immobilization [19], our results showed a decreased substrate conversion percentages using low substrate concentrations. This might be explained by mass-transfer limitations of the substrate to contact the enzyme [22] especially as the applied Ni-NTA sepharose is a gel-like matrix.…”
Section: Discussioncontrasting
confidence: 52%
“…Furthermore, dCK activity is essential for the activation of a wide variety of important therapeutic prodrugs, such as the antiviral agent lamivudine (3TC), for the treatment of HIV infection [16], or antineoplastic agents like cladribine [17] (Scheme 1). Both whole immobilized cells and purified enzymes have been used for the synthesis of NMP analogues [18,19]. Immobilization techniques help to stabilize biocatalysts under harsh conditions (e.g., high temperature, extreme pH, solvents) [19].…”
Section: Introductionmentioning
confidence: 99%
“…Both whole immobilized cells and purified enzymes have been used for the synthesis of NMP analogues [18,19]. Immobilization techniques help to stabilize biocatalysts under harsh conditions (e.g., high temperature, extreme pH, solvents) [19]. As the immobilized biocatalysts can be used repeatedly, the processes get more cost-effective, which is an important consideration for industrial applications.…”
Section: Introductionmentioning
confidence: 99%
“…Nickel-nitrilotriacetic acid (Ni-NTA) superflow was applied as the support, since the applied enzymes bear an N-terminal His-tag and it is described as a gentle way of immobilization. Both whole immobilized cells and purified enzymes have been used for the synthesis of NMP analogues [18,19]. Immobilization techniques help to stabilize biocatalysts under harsh conditions (e.g., high temperature, extreme pH, solvents) [19].…”
Natural ribonucleoside-5’-monophosphates are building blocks for nucleic acids which are used for a number of purposes, including food additives. Their analogues, additionally, are used in pharmaceutical applications. Fludarabine-5´-monophosphate, for example, is effective in treating hematological malignancies. To date, ribonucleoside-5’-monophosphates are mainly produced by chemical synthesis, but the inherent drawbacks of this approach have led to the development of enzymatic synthesis routes. In this study, we evaluated the potential of human deoxycytidine kinase (HsdCK) as suitable biocatalyst for the synthesis of natural and modified ribonucleoside-5’-monophosphates from their corresponding nucleosides. Human dCK was heterologously expressed in E. coli and immobilized onto Nickel-nitrilotriacetic acid (Ni-NTA) superflow. A screening of the substrate spectrum of soluble and immobilized biocatalyst revealed that HsdCK accepts a wide range of natural and modified nucleosides, except for thymidine and uridine derivatives. Upon optimization of the reaction conditions, HsdCK was used for the synthesis of fludarabine-5´-monophosphate using increasing substrate concentrations. While the soluble biocatalyst revealed highest product formation with the lowest substrate concentration of 0.3 mM, the product yield increased with increasing substrate concentrations in the presence of the immobilized HsdCK. Hence, the application of immobilized HsdCK is advantageous upon using high substrate concentration which is relevant in industrial applications.
The structurally unique "fleximer" nucleosides were originally designed to investigate how flexibility in an ucleobase could potentially affect receptor-ligand recognition and function. Recently they have been shown to have low-to-sub-micromolar levels of activity against an umber of viruses, including coronaviruses, filoviruses, and flaviviruses.H owever,t he synthesis of distal fleximersi np articularh as thus far been quite tedious and low yielding. As ap otential solution to this issue, as eries of proximal fleximer bases (flex-bases) has been successfully coupled to both ribose and 2'-deoxyribose sugars by using the N-deoxyribosyltransferase II of Lactobacillus leichmannii (LlNDT) and Escherichia coli purinen ucleoside phosphorylase (PNP). To explore the range of this facile approach, transglycosylation experiments on at hieno-expanded tricyclic heterocyclicb ase, as well as several distal and proximal flex-bases were performed to determine whether the corresponding fleximer nucleosides could be obtained in this fashion,t hus potentially significantly shortening the route to theseb iologically significant compounds.T he results of those studies are reported herein.
Nucleoside-2 ′-deoxyribosyl-transferases (NDTs) catalyze a transglycosylation reaction consisting of the exchange of the 2 ′-deoxyribose moiety between a purine and/or pyrimidine nucleoside and a purine and/or pyrimidine base. Because NDTs are highly specific for 2 ′-deoxyribonucleosides they generally display poor activity on modified C2 ′ and C3 ′ nucleosides and this limitation hampers their applicability as biocatalysts for the synthesis of modified nucleosides. We now report the production and purification of a novel NDT from Archaeoglobus veneficus that is endowed with native ribosyltransferase activity and hence it is more properly classified as an N-ribosyltransferase (AvNRT). Biophysical and biochemical characterization revealed that AvNRT is a homotetramer that displays maximum activity at 80 • C and pH 6 and shows remarkably high stability at high temperatures (60-80 • C). In addition, the activity of AvNRT was found to increase up to 2-fold in 4 M NaCl aqueous solution and to be retained in the presence of several water-miscible organic solvents. For completeness, and as a proof of concept for possible industrial applications, this thermophilic and halotolerant biocatalyst was successfully employed in the synthesis of different purine ribonucleoside analogs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.