Abstract
Due to the dual cofactor specificity, glucose 1-dehydrogenase (GDH) has been considered as a promising alternative for coenzyme regeneration in biocatalysis. To mine for potential GDHs for practical applications, several genes encoding for GDH had been heterogeneously expressed in Escherichia coli BL21 (DE3) for primary screening. Of all the candidates, GDH from Bacillus sp. ZJ (BzGDH) was one of the most robust enzymes. BzGDH was then purified to homogeneity by immobilized metal affinity chromatography and characterized biochemically. It displayed maximum activity at 45 °C and pH 9.0, and was stable at temperatures below 50 °C. BzGDH also exhibited a broad pH stability, especially in the acidic region, which could maintain around 80% of its initial activity at the pH range of 4.0–8.5 after incubating for 1 hour. Molecular dynamics simulation was conducted for better understanding the stability feature of BzGDH against the structural context. The in-silico simulation shows that BzGDH is stable and can maintain its overall structure against heat during the simulation at 323 K, which is consistent with the biochemical studies. In brief, the robust stability of BzGDH made it an attractive participant for cofactor regeneration on practical applications, especially for the catalysis implemented in acidic pH and high temperature.
Highlights
NAD(P)-dependent glucose 1-dehydrogenase (GDH, EC 1.1.1.47) is an oxidoreductase present in various organisms and involved in glucose metabolic pathways, catalyzing the oxidation of D-glucose to D-glucono-1,5-lactone while simultaneously reducing NAD(P) to NAD(P)H [1,2,3,4,5,6]
Analysis of the trajectory shows that BzGDH is stable and can maintain its overall structure against heat during the simulation at 323 Kelvin (K), which is in accordance with the biochemical studies
Amino acid substitutions mostly occurred at the N-terminus of GDHs (Figure 1), indicating that the N-terminal sequence is less conservative than the C-terminal sequence, which played critical roles in substrate recognition
Summary
NAD(P)-dependent glucose 1-dehydrogenase (GDH, EC 1.1.1.47) is an oxidoreductase present in various organisms and involved in glucose metabolic pathways, catalyzing the oxidation of D-glucose to D-glucono-1,5-lactone while simultaneously reducing NAD(P) to NAD(P)H [1,2,3,4,5,6]. Because many kinds of industrial enzymes are cofactor-dependent, the enzymatic synthesis is limited by the considerable expenses of the cofactors. To tackle the issue of manufacturing expense on biocatalysis, several cofactor regeneration approaches have been proposed, of which the enzymatic regeneration method has been considered as an effective technique [16]. Due to the activity toward both NAD and NADP, GDH has been proposed as a promising candidate for coenzyme regeneration [17,18], compared with other oxidoreductases such as formate dehydrogenase [19], alcohol dehydrogenase [20], glucose-6-phosphate dehydrogenase [21], and phosphite dehydrogenase [22]
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