Elsevier

Journal of Biotechnology

Volume 323, 10 November 2020, Pages 166-173
Journal of Biotechnology

Biotransformation of cladribine by a nanostabilized extremophilic biocatalyst

https://doi.org/10.1016/j.jbiotec.2020.08.012Get rights and content

Highlights

  • Thermomonospora alba CECT 3324 was selected for cladribine biosynthesis.

  • Whole cell entrapment in a novel nanostabilized hydrogel was achieved.

  • Operational stability of the novel biocatalyst was retained for 270 h.

  • Microscale assay allowed the production of 181 mg of cladribine at 30 °C.

Abstract

Cladribine (2-chloro-2′-deoxy-β-d-adenosine) is a 2′-deoxyadenosine analogue, approved by the FDA for the treatment of hairy cell leukemia and more recently has been proved for therapeutic against many autoimmune diseases as multiple sclerosis. The biosynthesis of this compound using Thermomonospora alba CECT 3324 as biocatalyst is herein reported. This thermophilic microorganism was successfully entrapped in polyacrylamide gel supplemented with nanoclays such as bentonite. The immobilized biocatalyst (T. alba-Ac-Bent 1.00 %), was able to biosynthesize cladribine with a conversion of 89 % in 1 h of reaction and retains its activity for more than 270 reuses without significantly activity loss, showing better operational stability and mechanical properties than the natural matrix. A microscale assay using the developed system, could allow the production of at least 181 mg of cladribine in successive bioprocesses.

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).

References (43)

  • C.W. Rivero et al.

    Biosynthesis of anti-HCV compounds using thermophilic microorganisms

    Bioorg. Med. Chem. Lett.

    (2012)
  • C.W. Rivero et al.

    Bioproduction of ribavirin by green microbial biotransformation

    Process Biochem.

    (2015)
  • N. Sundaraganesan et al.

    Vibrational spectra, assignments and normal coordinate calculation of acrylamide

    Talanta.

    (2001)
  • X. Zhou et al.

    Synthesis of 2,6-dihalogenated purine nucleosides by thermostable nucleoside phosphorylases

    Adv. Synth. Catal.

    (2015)
  • E. Adışen et al.

    Cladribine is a promising therapy for xanthoma disseminatum

    Clin. Exp. Dermatol.

    (2017)
  • B. Benli et al.

    Rheological, electrokinetic, and morphological characterization of alginate–bentonite biocomposites

    J. Appl. Polym. Sci.

    (2011)
  • V.A. Cappa et al.

    Bioproduction of floxuridine using nanostabilized biocatalysts

    Chem. Eng. Technol.

    (2016)
  • E.W. Co et al.

    The Development and Manufacture of Azacitidine, Decitabine, and Cladribine: Stereoselective Ribonucleoside Drug Synthesis Using the Vorbrüggen Glycosylation, Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage Process Development Volume 2

    (2016)
  • E.C. De Benedetti et al.

    Biotransformation of 2,6‐diaminopurine nucleosides by immobilized Geobacillus stearothermophilus

    Biotechnol. Progr.

    (2012)
  • M.D. Erion et al.

    Purine nucleoside phosphorylase

    2. Catalytic mechanism, Biochemistry

    (1997)
  • A. Fresco-Taboada et al.

    New insights on nucleoside 2′-deoxyribosyltransferases: a versatile biocatalyst for one-pot one-step synthesis of nucleoside analogs

    Appl. Microbiol. Biot.

    (2013)
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