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, 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.

Rational Design of a Thermostable 2′-Deoxyribosyltransferase for Nelarabine Production by Prediction of Disulfide Bond Engineering Sites

“…The use of NDTs as biocatalysts for the synthesis of NAs has been extensively described [ 6 , 9 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ]. However, even though NDT-mediated transglycosylation emerges as a versatile tool for industrial manufacturing of NAs, the long reaction times (24–48 h) required for the synthesis of some NAs (2′ and 3′ modified nucleosides) usually compromise the enzyme lifespan.…”

Programmable Polyproteams of Tyrosine Ammonia Lyases as Cross-Linked Enzymes for Synthesizing p-Coumaric Acid

“…So far, immobilization technology based on physical adsorption [ 10 ] (such as entrapment [ 11 ]) and covalent attachment [ 12 , 13 , 14 ] (such as crosslinking [ 15 ]) has matured [ 16 ] and been widely applied in the fields of the pharmaceutical industry, food industry, and wastewater treatment. In fact, the ligation intensity between enzymes and their supporting carriers resulting from physical adsorption is far weaker than that from covalent attachment.…”

“…Carrier-free immobilized enzyme(s), such as cross-linked dissolved enzymes (CLEs) and cross-linked enzyme crystals (CLECs) [ 17 , 18 ] provide a higher content of enzyme reactivity with higher space-time yields than carrier-bound enzymes, without taking into account the biocompatibility, toxicity, and cost of any supporting materials [ 19 , 20 , 21 ]. One prime example is glutaraldehyde mediated cross-linking [ 14 , 22 , 23 ], which is characterized by its low cost, high reactivity, and convenient operation and use. However, the random fixation that occurs in the glutaraldehyde coupling method often forms random polymorphisms that may harm the enzyme protein, leading to a decrease in enzyme activity.…”

Magnetic Multi-Enzymatic System for Cladribine Manufacturing

“…Due to its broad clinical applicability, the development of novel, efficient and eco-friendly alternatives to classical chemical methods are gaining ground. Among them, bioprocesses based on enzymatic transglycosylation from a nucleoside donor to a nucleobase acceptor catalyzed by 2′-deoxyribosyltransferases (NDTs), or nucleoside phosphorylases (NPs), are the most habitual strategies [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ]. Of these, we focused our attention on NDTs.…”