RESUMO
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, SiGPEI 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, SiGPEI 25000-LdNDT-Alg was successfully employed in the green production of cladribine at mg scale.
Assuntos
Cladribina/metabolismo , Lactobacillus delbrueckii/enzimologia , Transferases/química , Transferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glutaral/química , Química Verde , Concentração de Íons de Hidrogênio , Dióxido de Silício/química , TemperaturaRESUMO
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.
Assuntos
Biotransformação , Cladribina/metabolismo , Extremófilos/fisiologia , Resinas Acrílicas , Antineoplásicos/uso terapêutico , Vias Biossintéticas , Cladribina/uso terapêutico , Desoxiadenosinas , Geobacillus , Leucemia de Células Pilosas/tratamento farmacológico , Nanocompostos , Temperatura , Thermobifida/crescimento & desenvolvimento , Thermobifida/metabolismoRESUMO
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.
Assuntos
Antineoplásicos/metabolismo , Células Imobilizadas/metabolismo , Cladribina/metabolismo , Lactobacillus/metabolismo , Alginatos/química , Proteínas de Bactérias/metabolismo , Biotransformação , Dimetil Sulfóxido/farmacologia , Lactobacillus/efeitos dos fármacos , Imãs , Pentosiltransferases/metabolismoRESUMO
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.
Assuntos
Alginatos/química , Antineoplásicos/metabolismo , Cladribina/metabolismo , Micrococcaceae/metabolismo , Antineoplásicos/química , Biocatálise , Biotransformação , Cladribina/químicaRESUMO
Chlorinase SalL halogenate S-adenosyl-l-methionine (SAM) reacts with chloride to generate 5'-chloro-5'-deoxyadenosine and l-methionine through a nucleophilic substitution mechanism. Although it is known that chlorinase enhances the rate of reaction by a factor of 1.2 × 1017 fold, it is not entirely clear how this is accomplished. The search for the origin of the catalysis of chlorinase and other enzymes has led to a desolvation hypothesis. In the present work, we have used well defined computational simulations in order to evaluate the origin of the catalytic efficiency of chlorinase. The results demonstrate that the catalytic effect of chlorinase is associated with the fact that Cl- is "solvated" by the protein more than by the reference solution reaction, which is not in accordance with proposed catalysis by desolvation. It is found that chlorinase SalL active sites provide electrostatic stabilization of the transition state which is the origin of its catalytic effect.