Bioproduction of ribavirin by green microbial biotransformation

Abstract

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.

Keywords: Antiviral agent; Biocatalysis; Immobilization; Scale-up.

Graphical abstract

Scheme 1

Scheme of reaction for ribavirin biotransformation using whole cells. In this work, uridine (Urd) and 1 H-[1,2,4] triazole-3-carboxamide (TCA) were used as ribose donor and modified base, respectively. UP: uridine phosphorylase and PNP: purine nucleoside phosphorylase.

Fig. 1

Optimization of the amount of microorganisms for ribavirin biotransformation. Reaction was performed in sodium phosphate buffer (30 mM, pH 7) containing 2.5 mM Urd and TCA at 60 °C and 200 rpm with 5 × 10⁸ CFU (•), 1 × 10⁹ CFU (○), 5 × 10⁹ CFU (▴), 1 × 10¹⁰ CFU (♢), 5 × 10¹⁰ CFU (■). All reactions were performed three times and bioconversion percentage was calculated as: (mmol product/mmol limiting reagent) × 100.

Fig. 2

A) Optimization of reaction temperature for ribavirin bioconversion. Biotransformation was performed during 1 h using 2.5 mM Urd and TCA in sodium phosphate buffer (30 mM, pH 7) at different temperatures and 200 rpm. According to Duncan test for data media comparison, significant differences among treatments named with different letter (p-value < 0.05) were observed. Productivity was calculated as: mmol ribavirin/L × h. B) Biotransformation was evaluated using different amounts of substrates, 10 mM TCA: 2.5 mM Urd (○); 2.5 mM TCA: 2.5 mM Urd (□); 2.5 mM TCA: 10 mM Urd (▴) at 30 °C and 200 rpm. All reactions were performed three times and bioconversion percentage was calculated as: (mmol product/mmol limiting reagent) × 100.

Fig. 3

Ribavirin biotransformation was evaluated at different growth stages. Reactions were performed during 3 h at 30 °C and 200 rpm, using 10 mM TCA and 2.5 mM Urd in sodium phosphate buffer (30 mM, pH 7). According to Duncan test for data media comparison, significant differences among treatments named with different letter (p-value < 0.05) were observed. All reactions were performed three times and bioconversion rate was calculated as: (mmol product/mmol limiting reagent) × 100.

Fig. 4

Storage stability assay for E. coli immobilized in agarose 3% (w/v) (•) and polyacrylamide 20% (w/v) (▴). Reactions were performed three times at optimal conditions (10 mM TCA and 2.5 mM Urd, at 30 °C and 200 rpm during 3 h). Relative activity was calculated using the initial condition as control.