Jorge Abel Trelles

Nowadays, enzyme-mediated processes offer an eco-friendly and efficient alternative to the traditional multistep and environmentally harmful chemical processes. Herein we report the enzymatic synthesis of cladribine by a novel 2′-deoxyribosyltransferase (NDT)-based combined biocatalyst. To this end, Lactobacillus delbrueckii NDT (LdNDT) was successfully immobilized through a two-step immobilization methodology, including a covalent immobilization onto glutaraldehyde-activated biomimetic silica nanoparticles followed by biocatalyst entrapment in calcium alginate. The resulting immobilized derivative, SiG_{PEI 25000}-LdNDT-Alg, displayed 98% retained activity and was shown to be active and stable in a broad range of pH (5–9) and temperature (30–60 °C), but also displayed an extremely high reusability (up to 2100 reuses without negligible loss of activity) in the enzymatic production of cladribine. Finally, as a proof of concept, SiG_{PEI 25000}-LdNDT-Alg was successfully employed in the green production of cladribine at mg scale.

A novel IDA-LaNDT derivative was able to reach the highest productivity in the biosynthesis of a well-known antitumoral agent called decitabine. However, the combination of two simple and inexpensive techniques such as ionic absorption and gel entrapment with the incorporation of a bionanocomposite such as bentonite significantly improved the stability of this biocatalyst. These modifications allowed the enhancement of storage stability (for at least 18 months), reusability (400 h of successive batches without significant loss of its initial activity), and thermal and solvent stability with respect to the non-entrapped derivative. Moreover, reaction conditions were optimized by increasing the solubility of 5-aza by dilution with dimethylsulfoxide. Therefore, a scale-up of the bioprocess was assayed using the developed biocatalyst, obtaining 221 mg/L·h of DAC. Finally, green parameters were calculated using the nanostabilized biocatalyst, whose results indicated that it was able to biosynthesize DAC by a smooth, cheap, and environmentally friendly methodology.

In the present study, a novel extracellular laccase isolated from Geobacillus stearothermophilus ATCC 10149 was entrapped in a bionanocomposite matrix consisting of copper alginate (Cu-alginate) supplemented with the nanoclay bentonite. After optimization, this nanobiocatalyst was able to degrade up to 90% of Remazol Brilliant Blue R (RBBR) without the addition of redox mediators and retained 70% of its initial activity for at least 1440 h, equivalent to more than 288 uses. The incorporation of nanoclay allowed alginate beads to be used in alkaline pH and strengthened its mechanical properties. Besides, this thermophilic laccase was able to decolorize other structurally different synthetic dyes such as Methyl Orange, Malachite Green and Indigo Carmine. These preliminary results suggested that the nanobiocatalyst could be a suitable option for dye decolorization and be further developed for large scale bioremediation of toxic dyes.

A stable biocatalyst with magnetic properties based on immobilized Lactobacillus animalis ATCC 35,046 to obtain 2-chloroadenine-2′-deoxyriboside, known as cladribine, is reported for the first time. This nucleoside analogue is an antitumor agent used in the treatment of a wide variety of types of leukemia. In this study, an eco-compatible and alternative bioprocess to obtain cladribine was developed. Product conversion was close to 90% at 2 h in optimized nonconventional reaction media. The microscale biosynthesis of the compound of interest afforded a total productivity close to 370 mg/L/h in the presence of DMSO, and it was stable at least for 30 days in storage conditions.

Microbial whole cells are efficient, ecological, and low-cost catalysts that have been successfully applied in the pharmaceutical, environmental, and alimentary industries, among others. Microorganism immobilization is a good way to carry out the bioprocess under preparative conditions. The main advantages of this methodology lie in their high operational stability, easy upstream separation, and bioprocess scale-up feasibility. Cell entrapment is the most widely used technique for whole cell immobilization. This technique—in which the cells are included within a rigid network—is porous enough to allow the diffusion of substrates and products, protects the selected microorganism from the reaction medium, and has high immobilization efficiency (100% in most cases).

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.