Enzymatic characterization of a thermostable 6-phosphogluconate dehydrogenase from Hydrogenobacter thermophilus and its application for NADH regeneration.

Abstract

6-Phosphogluconate dehydrogenases (6PGDHs) are widely existing as reduced cofactor (NADH/NADPH) regeneration biocatalysts. Herein, a thermostable 6PGDH from Hydrogenobacter thermophilus (Ht6PGDH) was overexpressed in Escherichia coli and enzymologically characterized. Ht6PGDH exhibited exceptional stability and catalytic activity under high-temperature conditions, with an optimum temperature of 85 °C and the ability to maintain high activity for prolonged periods at 70 °C, which could be purified through a one-step heat treatment. Moreover, Ht6PGDH exhibited a preference for NAD⁺ with a K _m value of 0.4 mM and a k _cat value of 28.6 s⁻¹, demonstrating a significant preference over NADP⁺. These properties render Ht6PGDH a potentially valuable enzyme for high-temperature bioconversion and in vitro synthetic biosystems. Additional research showed that Ht6PGDH excelled in the regeneration of NADH, achieving efficient lactate production when integrated into an in vitro synthetic biosystem containing lactate dehydrogenase (LDH). Furthermore, the cascade reaction of Ht6PGDH with glucose-6-phosphate dehydrogenase (G6PDH) was explored for NADH regeneration using starch as the substrate, further validating its potential application in complex biosynthetic systems.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-024-04165-6.