One-Pot, One-Step Production of Dietary Nucleotides by Magnetic Biocatalysts

Abstract

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 • C during 24 h; 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.

Green Production of Cladribine by Using Immobilized 2′-Deoxyribosyltransferase from Lactobacillus delbrueckii Stabilized through a Double Covalent/Entrapment Technology

“…In contrast, covalent immobilization onto SiG nanoparticles or CNBr agarose beads displayed negligible effects on enzyme activity. According to Table S1 , a pKa = 7 is deduced for N-terminus, which is lined with an unipunctual immobilization through N-terminus under immobilization conditions required for immobilization in SiG nanoparticles or CNBr agarose (pH 7.0) [ 48 , 49 , 50 , 51 ]. Moreover, as shown in Table S1 , Lys 48 and 62 residues are not deprotonated at pH 7.0, which prevents the immobilization through these surface Lys residues, which avoids the distortion of the active site architecture [ 6 , 7 ].…”

“…In contrast, covalent immobilization onto SiG nanoparticles or CNBr agarose beads displayed negligible effects on enzyme activity. According to Table S1, a pKa = 7 is deduced for N-terminus, which is lined with an unipunctual immobilization through N-terminus under immobilization conditions required for immobilization in SiG nano- In this context, results reported by the H++ protonation predictor program (http: //biophysics.cs.vt.edu/H++, accessed on 24 March 2021) (Table S1, Supplemental Material) [48][49][50] displayed that both, Lys 48 and 62, are deprotonated at pH values required for immobilization on glyoxyl agarose (pH 10-11). As a consequence, they can be involved in the covalent linkages, reducing the mobility and flexibility of this catalytic loop and avoiding the proper binding orientation of pyrimidine ring during the catalytic process, which leads to a pronounced loss of activity.…”

“…In this context, results reported by the H++ protonation predictor program ( , accessed on 24 March 2021) ( Table S1, Supplemental Material ) [ 48 , 49 , 50 ] displayed that both, Lys 48 and 62, are deprotonated at pH values required for immobilization on glyoxyl agarose (pH 10–11). As a consequence, they can be involved in the covalent linkages, reducing the mobility and flexibility of this catalytic loop and avoiding the proper binding orientation of pyrimidine ring during the catalytic process, which leads to a pronounced loss of activity.…”

Immobilized Biocatalysts

“…According to Míguez et al [30], a recombinant β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous that was expressed in Pichia pastoris turned out to be well suited for the synthesis of various neo-fructooligosaccharides such as neokestose, 1-kestose, neonystose, and blastose in a batch reactor at 30 • C. The β-fructofuranosidase was immobilized in polyvinyl alcohol lenticular particles, and could be reused for at least seven cycles without loss of activity. The tetrameric hypoxanthine-guanine-xanthine phosphoribosyltransferase from Thermus thermophilus HB8 was immobilized by del Arco et al [31] onto glutaraldehyde-activated MagReSyn ® Amine magnetic iron-oxide porous microparticles (MTtHGXPRT) by different strategies for the one-pot, one-step production of dietary nucleotides. The best variant showed negligible loss of activity at 60 • C during 24 h, and a reusability of up to seven cycles.…”

Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5′-Mono-, Di- and Triphosphates

“…6-oxopurine phosphoribosyltransferases (6-oxo PRTs, EC 2.4.2.8, EC 2.4.2.22) are essential enzymes in the purine salvage pathway ( el Kouni, 2003 ). 6-oxo PRTs catalyze the reversible transfer of the 5-phosphoribosyl group from 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP) to N9 on the 6-oxopurine bases hypoxanthine (1), guanine (2) or xanthine (3) to form IMP (4), GMP (5) or XMP (6) (HPRT, GPRT, XPRT, HGPRT, GXPRT or HGXPRT), respectively, in the presence of Mg 2+ ( Del Arco et al, 2017 , 2018c ; Figure 1A ).…”