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

Abstract

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.

Keywords: antineoplastic drug; biomimetic silica; calcium alginate; entrapment; enzyme immobilization; glutaraldehyde.

Figure 1

Schematic representation of the combined immobilization procedure, carried out in this work to obtain cladribine, through the development of a highly stabilized biocatalyst. TMOS: tetramethylorthosilicate, PEI: Polyethyleneimine and LdNDT: Recombinant NDT from Lactobacillus delbrueckii.

Figure 2

Overall representation of surface exposed Lys residues (atom colored sticks) in LdNDT active site complexed with Thd (atom colored sticks). The cartoon representation is used for simplicity and ease of visualization. The catalytic loop region (amino acids 48 up to 62) is highlighted in blue. The figure has been prepared with PyMOL [34].

Figure 3

Study of several parameters using SiG_PEI25000-LdNDT derivative for cladribine biosynthesis. (A) Effect of pH on reaction time course, (?) 25 mM sodium acetate buffer, pH 5.0; (•) 25 mM tris-HCl buffer, pH 7.0; (▲) 25 mM tris-HCl buffer, pH 9.0. (B) Effect of temperature on reaction time course, (•) 30 °C, (?) 50 °C, and (▲) 60 °C.

Figure 4

Thermal inactivation of SiG_PEI25000-LdNDT (A) and SiG_PEI25000-LdNDT-Alg (B) at different temperatures (▲) 30 °C, (□) 50 °C, and (○) 60 °C. Effect of pH on the stability of SiG_PEI25000-LdNDT (C) and SiG_PEI25000-LdNDT-Alg (D). pH 5.0 (▲), pH 7.0 (□), and pH 9.0 (○).

Figure 5

Surface representation of hexamericLdNDT. The different chains (A–F) are highlighted in different colors. N-terminus residues are yellow colored.

Figure 6

SEM and EDX analysis of SiG_PEI25000-LdNDT (A,C) and SiG_PEI25000-LdNDT-Alg (B,D) biocatalysts.

Figure 7

Storage stability at 4 °C (A) and reusability (B) of (▲)SiG_PEI25000-LdNDT-Alg, (□) SiG_PEI25000-LdNDT, and (○) CNBr-LdNDT.