Glucose-6-phosphate dehydrogenase immobilized onto magnetic beads (G6PDH-Mb) as a generator system for production of NADPH: Development and application in metabolism studies

Autor(es)

Furlani, Izadora Liranco ; Oliveira, Regina Vincenzi ; Cass, Quezia Bezerra

• Tipo de documento: Artigo Científico
• Fonte: Journal of Pharmaceutical and Biomedical Analysis; v. 219, p. 9-pg., 2022-06-30.

Resumo

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) participates in several anabolic and catabolic pathways, being essential in numerous biochemical reactions involving energy release. Most of these reactions require a high amount of NADPH, which can be expensive from an industry point of view. Thus, biotechnology industries developed a great interest in NADPH production. Currently, there are different ways to obtain NADPH in situ, however, the most common is by enzymatic reactions, known as generator systems. Although this approach can be beneficial in terms of cost, the major drawback is the impossibility of reusing the enzyme. To overcome this, enzyme immobilization is a proven alternative. Herein, we report the use of glucose-6-phosphate dehydrogenase immobilized onto magnetic beads (G6PDH-Mb) through glutaraldehyde coupling to produce high amounts of NADPH. The G6PDH-Mbs were kinetically characterized showing a sigmoidal curve. Besides, the stability was evaluated, and their reuse was demonstrated for a period superior to 40 days. The G6PDH-Mb was used to in situ production of the NADPH metabolism experiments, using human liver microsome solutions and either albendazole or fiscalin B as model targets. The production of in vitro metabolites from albendazole and fiscalin B was evaluated by comparing the use of NADPH generated in situ with those obtained by commercial NADPH. Moreover, the activity of the G6PDH-Mb was monitored after using it for five consecutive albendazole metabolism reactions, with only a minor decrease in the enzyme activity (3.58 +/- 1.67%) after the fifth time of use. The higher concentration obtained when using the designed G6PDH-Mb generator system demonstrated proof of the concept and its applicability. (AU)

• Processo FAPESP: 20/05965-8 - Estratégias metabolômicas baseadas em LC-HRMS para investigação de potenciais biomarcadores para diagnóstico clínico - Fase I
• Beneficiário: Regina Vincenzi Oliveira
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 14/50249-8 - Green chemistry: sustainable synthetic methods employing benign solvents, safer reagents, and bio-renewable feedstock
• Beneficiário: Arlene Gonçalves Corrêa
• Modalidade de apoio: Auxílio à Pesquisa - Programa Centros de Pesquisa em Engenharia
• Processo FAPESP: 18/03035-3 - Desenvolvimento de biorreatores para estudos online do metabolismo de fármacos
• Beneficiário: Izadora Liranço Furlani
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto
• Processo FAPESP: 14/50244-6 - UHPLC acoplado a espectrômetro de massa do tipo híbrido com analisador quadrupolar e time of flight
• Beneficiário: Quezia Bezerra Cass
• Modalidade de apoio: Auxílio à Pesquisa - Programa Equipamentos Multiusuários