Cloning, Expression and Characterization of a Highly Active Alcohol Dehydrogenase for Production of Ethyl (S)-4-Chloro-3-Hydroxybutyrate

Abstract

A novel alcohol dehydrogenase from Bartonella apis (BaADH) was heterologous expressed in Escherichia coli. Its biochemical properties were investigated and used to catalyze the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is a chiral intermediate of the cholesterol-lowering drug atorvastatin. The purified recombinant BaADH displayed 182.4 U/mg of the specific activity using ethyl 4-chloroacetoacetate as substrate under the conditions of 50°C in pH 7.0 Tris-HCl buffer. It was stable in storage buffers of pH 7 to 9 and retains up to 96.7% of the initial activity after 24 h. The K m and V max values of BaADH were 0.11 mM and 190.4 lmol min-1 mg-1 , respectively. Synthesis of (S)-CHBE catalyzed by BaADH was performed with a cofactor regeneration system using a glucose dehydrogenase, and a conversion of 94.9% can be achieved after 1 h reaction. Homology modeling and substrate docking revealed that a typical catalytic triad is in contact with local water molecules to form a catalytic system. The results indicated this ADH could contribute to the further enzymatic synthesis of (S)-CHBE.

Formation of Unstable and very Reactive Chemical Species Catalyzed by Metalloenzymes: A Mechanistic Overview

“…Zinc cofactors can also catalyze hydride transfers. This type of reaction can, for example, be found in alcohol dehydrogenases (EC 1.1.1.1, ADH), a group of dehydrogenase enzymes that facilitates the interconversion between alcohols, ketones, and aldehydes through the reduction of nicotinamide derivatives NAD + or NADP + to the corresponding 1,4-dihydronicotinamides [139,140,141,142]. This role is particularly important in humans and other animals, to degrade alcohols that otherwise would be toxic for the cells.…”

One‐step selective affinity purification and immobilization of His‐tagged enzyme by recyclable magnetic nanoparticles

“…It mainly was used for specifically conversion from β-d-glucose to β-dgluconolactone using NAD(P) + as a cofactor, and also can be used in diagnostic for clinical blood glucose measurement [36,37], electrochemical detection [38], as well as biofuel cell [39,40]. Besides, it can be employed for cofactor regeneration [41]. Despite the wide range of applications and interests, the free glucose dehydrogenase has some inevitable limitations such as complex purification process, unrecyclable, and difficult to separate from product.…”

Identification of a newly isolated <i>Sphingomonas</i> sp. LZ1 and its application to biosynthesize chiral alcohols

“…Various strategies, such as chromatographic separation, enantioselective resolution or dynamic kinetic resolution, and asymmetric reduction, have been developed for the synthesis of chiral alcohols (Zheng et al, 2017). Asymmetric biocatalytic reduction of ketones is an efficient and most powerful way to prepare optically pure alcohols because of its high enantioselectivity, 100% theoretical yield, and environmentally friendly and meeting green chemistry guidelines (Luo et al, 2018;Zhu et al, 2019). Many microorganisms or isolated enzymes have been applied as biocatalysts in the asymmetric biocatalytic reduction process (Shah et al, 2018;Wang et al, 2017;Wu et al, 2015).…”