Biotransformation of cladribine using a stabilized biocatalyst in calcium alginate beads

Abstract

Cladribine is a nucleoside analogue widely used in the pharmaceutical industry for the treatment of several neoplasms, including hairy‐cell leukemia among others. This compound has also shown efficacy in the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In this work, a green bioprocess for cladribine biosynthesis using immobilized Arthrobacter oxydans was developed. The microorganism was stabilized by entrapment immobilization in the natural matrix alginate. Different reaction parameters were optimized obtaining a biocatalyst able to achieve cladribine bioconversion values close to 85% after 1 hr, the shortest reaction times reported so far. The developed bioprocess was successfully scaled‐up reaching a productivity of 138 mg L−1 hr−1. Also, the biocatalyst was stable for 5 months in storage and in 96 hr at operational conditions.

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

“…The double stabilized biocatalyst was able to achieve 86% of cladribine conversion at 15 min, while after 30 min of reaction a conversion of 96% was achieved, equaling that achieved by its counterpart without trapping. As expected, the biosynthetic rate of the entrapped biocatalyst decreased slightly, probably due to diffusion restrictions associated with the alginate coating [ 54 , 55 , 56 ].…”

“…With the aim of optimizing the biocatalyst for future scale-up studies, SiG PEI 25000 - Ld NDT derivative was successfully entrapped in calcium alginate to obtain SiG PEI25000 - Ld NDT-Alg biocatalyst [ 29 , 54 , 55 , 56 ]. The double stabilized biocatalyst was able to achieve 86% of cladribine conversion at 15 min, while after 30 min of reaction a conversion of 96% was achieved, equaling that achieved by its counterpart without trapping.…”

“…Although a high number of NDT-mediated bioprocesses using immobilized biocatalysts have been reported during the last decade [ 10 , 11 , 29 , 32 , 33 , 38 , 42 , 43 , 45 , 46 , 54 , 55 , 60 , 61 ], most of them focused on cladribine synthesis [ 11 , 30 , 38 , 45 , 54 , 61 ], the low biocatalyst reusability under assay conditions often hinders their industrial implementation. Illustrative examples of this operational problem are cladribine synthesis using PDT from Trypanosoma brucei immobilized ( Tb PDT) onto glutaraldehyde-activated microparticles (25 reuses without any activity loss, 1 mM substrates, 41% conversion) [ 45 ] or mutant Tb PDT V11S immobilized onto Ni 2+ chelate magnetic microparticles (10 reuses without any activity loss, 2 mM substrates, 21% conversion) [ 61 ].…”

“…This operational problem was also observed for other immobilized enzymes (e.g., cascade synthesis of cladribine using immobilized PNP and PyNP from Geobacillus stearothermophilus ; 20 reuses, 5 mM substrates 85% conversion) [ 62 ]. Interestingly, several recent articles have addressed this issue, developing novel immobilized derivatives with high operational stability and reusability, such as Arthrobacter oxydans cells immobilized on alginate (20 reuses, 0.5 mM sub-strates, 85% conversion) [ 54 ] or Thermomonospora alba whole cells entrapped in nanostabilized hydrogels (270 reuses, 0.5 mM substrates, 89% conversion) [ 38 ]. However, these experimental results are surmounted by those shown in this work (2100 reuses, 1 mM, ≈95% conversion).…”