Biotransformation of cladribine by a nanostabilized extremophilic biocatalyst.

Cladribine (2-chloro-2'-deoxy-ß-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.

Biosynthesis of an antiviral compound using a stabilized phosphopentomutase by multipoint covalent immobilization.

Ribavirin is a synthetic guanosine analogue with a broad-spectrum of antiviral activity. It is clinically effective against several viruses, such as respiratory syncytial virus, several hemorrhagic fever viruses and HCV when combined with pegylated interferon-α. Phosphopentomutase (PPM) catalyzes the transfer of intramolecular phosphate (from C1 to C5) on ribose, and is involved in pentose phosphate pathway and in purine metabolism. Reactions catalyzed by this enzyme are useful for nucleoside analogues production. However, out of its natural environment PPM is unstable and its stability is affected by parameters such as pH and temperature. Therefore, to irreversibly immobilize this enzyme, it needs to be stabilized. In this work, PPM from Escherichia coli ATCC 4157 was overexpressed, purified, stabilized at alkaline pH and immobilized on several supports. The activity of different additives as stabilizing agents was evaluated, and the best result was found using 10% (v/v) glycerol. Under this condition, PPM maintained 86% of its initial activity at pH 10 after 18h incubation, which allowed further covalent immobilization of this enzyme on glyoxyl-agarose with a high yield. This is the first time that PPM has been immobilized by multipoint covalent attachment on glyoxyl support, this derivative being able to biosynthesize ribavirin from α-d-ribose-5-phosphate.

Bioproduction of ribavirin by green microbial biotransformation.

Ribavirin is an antiviral compound widely used in Hepatitis C Virus therapy. Biotransformation of this nucleoside analogue using Escherichia coli ATCC 12407 as biocatalyst is herein reported. Reaction parameters such as microorganism amounts, substrate ratio and temperature were optimized reaching conversion yields of 86%. Biocatalyst stability was enhanced by immobilization in agarose matrix. This immobilized biocatalyst was able to be reused for more than 270 h and could be stored during more than 4 months without activity loss. Batch and packed-bed reactors based on a stabilized biocatalyst were assayed for bioprocess scale-up. A continuous sustainable bioprocess was evaluated using a prototype packed-bed reactor, which allowed to produce 95 mg of ribavirin. Finally, in this work an efficient green bioprocess for ribavirin bioproduction using a stabilized biocatalyst was developed.

Biosynthesis of anti-HCV compounds using thermophilic microorganisms.

This work describes the application of thermophilic microorganisms for obtaining 6-halogenated purine nucleosides. Biosynthesis of 6-chloropurine-2'-deoxyriboside and 6-chloropurine riboside was achieved by Geobacillus stearothermophilus CECT 43 with a conversion of 90% and 68%, respectively. Furthermore, the selected microorganism was satisfactorily stabilized by immobilization in an agarose matrix. This biocatalyst can be reused at least 70 times without significant loss of activity, obtaining 379mg/L of 6-chloropurine-2'-deoxyriboside. The obtained compounds can be used as antiviral agents.

Biotransformation of 2,6-diaminopurine nucleosides by immobilized Geobacillus stearothermophilus.

An efficient and green bioprocess to obtain 2,6-diaminopurine nucleosides using thermophilic bacteria is herein reported. Geobacillus stearothermophilus CECT 43 showed a conversion rate of 90 and 83% at 2 h to obtain 2,6-diaminopurine-2'-deoxyriboside and 2,6-diaminopurine riboside, respectively. The selected biocatalyst was successfully stabilized in an agarose matrix and used to produce up to 23.4 g of 2,6-diaminopurine-2'-deoxyriboside in 240 h of process. These nucleoside analogues can be used as prodrug precursors or in antisense oligonucleotide synthesis.