Biotransformation of cladribine by a nanostabilized extremophilic biocatalyst
Introduction
Nucleosides are natural molecules with a key role in DNA biosynthesis. Their analogues are of interest in the pharmaceutical industry due to their broad spectrum of activity as antiviral, antitumor compounds or in autoimmune diseases (Mulakayala et al., 2013). These molecules could be produced by chemical processes or enzymatic modification of natural precursors (Xu et al., 2011; Lapponi et al., 2016).
Cladribine (2-chloro-2-deoxy-β-d-adenosine; 2-ChldAdo) is a halogenated deoxyadenosine analogue used in the treatment of hairy cell leukemia and indicated for relapsing forms of multiple sclerosis. In these cases, this drug is used in patients whose disease is highly active. This compound acts as an antimetabolite in lymphocytes and monocytes due to its resistance to deamination by human adenosine deaminase (EC: 3.5.4.4), which causes accumulation of 2-ChldAdo-triphosphate and further cell death by DNA repair interference. As a result of its selective lymphotoxic effects, this molecule has been tested for the treatment of many hematologic malignancies, such as leukemia, multiple sclerosis, rheumatologic diseases, and even rare malignancies (Spurgeon et al., 2009; Adışen et al., 2017).
Although several methods have been reported for cladribine biosynthesis, many refer to chemical reactions that require numerous steps of protection and deprotection of reactive groups, complex purification techniques for chiral subproduct removal, and afford low productivity yields (Peng, 2013).
Currently, biocatalytic processes are renowned as an environmentally friendly alternative for the biosynthesis of therapeutic agents. Nucleoside analogues can be obtained by transglycosilation reactions catalyzed by nucleoside phosphorylases (NPs) (Rivero et al., 2015) or 2′-N-deoxyribosyltransferases (NDTs) (Fresco-Taboada et al., 2013). These enzymatic biocatalysts can be used in their isolated form or contained in whole cells. The use of complete microorganisms allows one-pot reactions and extended enzymatic stability (Lin and Tao, 2017).
In particular, Thermomonospora alba is a thermophilic Gram-positive microorganism from the actinomycete family. Many related microorganisms have been studied as recognizable sources for enzymes with industrial interest (Shrestha et al., 2016) and as biocatalysts for obtaining nanoparticles (Manivasagan et al., 2016). Wild type biocatalysts can be easily cultured and stabilized by immobilization techniques, favoring biosynthetic processes in a single event with high yields at short reaction times (Cappa et al., 2014).
Immobilization techniques enable biocatalyst recovery and reusability, improving bioprocess productivity (De Benedetti et al., 2015). Entrapment methodologies are the most widely used for whole cell stabilization hydrogels, thermogels and synthetic polymers being the most common matrices. Polyacrylamide is formed by acrylamide subunits that can be cross-linked with N,N'-methylene-bisacrylamide, obtaining a tighter gel structure. The polymerization process can be easily performed and has been used for biocatalyst stabilization over the years (Zajkoska et al., 2013).
Although in some cases the polymer structure could be affected by biocatalytic reaction conditions, the addition of nanoclays such as bentonite could improve the mechanical properties of more widely used matrices (Cappa et al., 2016). The polymer-clay nanocomposites obtained by adding low amounts of bentonite to polyacrylamide are a new class of material used in biocatalyst stabilization. Therefore, an environmentally friendly bioprocess for Cladribine biosynthesis using a nanostabilized polyacrylamide matrix for Thermomonospora alba CECT 3324 immobilization was developed.
Section snippets
Reagents and microorganisms
Nucleosides were purchased from Sigma Chem. Co. (Brazil). HPLC grade solvents were supplied by Sintorgan S.A (Argentina). Culture medium components were purchased from Britania S.A. (Argentina). Microorganism strains belong to our own laboratory collection.
Growth conditions
Geobacillus strains were cultured at 55 °C and 200 rpm in medium containing 10 g/L meat peptone, 5 g/L yeast extract, 5 g/L NaCl, and 4 g/L glucose (pH 7.0). Thermomonospora and Streptomyces strains were cultured at 50 °C and 200 rpm, in
Screening
The ability of extremophiles to produce a wide variety of antitumor and antiviral compounds of industrial interest has been demonstrated in previous reports (Rivero et al., 2012). Thus, three genera of thermophilic microorganisms were tested for cladribine biosynthesis. Of the nine strains studied, three were active, two of which belong to the genus Geobacillus and the other to the genus Thermomonospora. The two Streptomyces strains tested for this nucleoside biosynthesis did not show catalytic
Conclusions
A thermophilic biocatalyst, Thermomonospora alba CECT 3324, was selected. This microorganism was able to biosynthesize cladribine with high yields at short reaction times. Furthermore, this microorganism was successfully entrapped in a novel nanostabilized hydrogel composed of polyacrylamide and bentonite. Clay addition to conventional polyacrylamide improved matrix hardness, immobilized biocatalyst stability and reusability. Finally, a scale-up of the bioprocess was assayed allowing the
CRediT authorship contribution statement
Cintia W. Rivero: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resourses, Writing - review & editing, Supervision, Funding acquisition. Eliana C. De Benedetti: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft. Jorge Sambeth: Formal analysis, Investigation, Resourses. Jorge A. Trelles: Conceptualization, Validation, Formal analysis, Resourses, Writing - review & editing, Supervision, Project administration, Funding
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
This research was supported by Agencia Nacional de Promoción Científica y Tecnológica (PICT 2013-2658 and PICT 2014-3438), Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2014-KA5-00805) and Universidad Nacional de Quilmes (PUNQ1036/11).
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Cell immobilization strategies for biotransformations
2022, Current Opinion in Green and Sustainable ChemistryCitation Excerpt :First, Arthrobacter oxydans was immobilized in calcium alginate for the bioproduction of the antineoplastic compound cladribine, achieving high bioconversion values at only 1 h of reaction with a reusability of 20 cycles [11]. Besides, in a later study, the same analog was bioproduced by Thermomonospora alba entrapped in the polyacrylamide gel supplemented with bentonite, achieving a biocatalyst with the 270-cycle reuse number, showing how the addition of nanocomposites improved biocatalyst properties [12]. In addition, cladribine was obtained by a biocatalyst with magnetic properties based on immobilized Lactobacillus animalis in alginate-iron filing.