Isolation and Biochemical Characterization of a Glucose Dehydrogenase from a Hay Infusion Metagenome

Abstract

Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P) as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P), the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.

Rapid Model-Based Optimization of a Two-Enzyme System for Continuous Reductive Amination in Flow

“…Importantly, these models are available through github and can be run using cloud-based python notebooks, making them very accessible. The Ad RedAm reaction was modeled as separate forward and reverse reactions using the three-substrate rate equation for Ad RedAm described previously ( r1 and r2 ). , The Bs GDH reaction was modeled as an irreversible two-substrate ordered sequential reaction, assuming the hydrolysis of glucono delta-lactone (GDL) drives the reaction in the forward direction ( r3 ). , To confirm whether a buildup of GDL might be an issue, the hydrolysis of GDL was also included in the model ( r4 ). In order to model the reaction in flow, the column was approximated as a homogeneous container, with the rate of dilution or addition of each compound into or out of the column modeled according the flow rate and column void volume ( r5 ).…”

“…5,46 The BsGDH reaction was modeled as an irreversible twosubstrate ordered sequential reaction, assuming the hydrolysis of glucono delta-lactone (GDL) drives the reaction in the forward direction (r3). 47,48 To confirm whether a buildup of Table 1. Mechanistic Model Rate Laws for r1 to r4…”

Enzymes of an alternative pathway of glucose metabolism in obligate methanotrophs

“…To date, microbial NAD(P) + -GDHs have been characterized in gram-positive bacteria, cyanobacteria, archaea, fungi and yeasts, as well as in plants, whereas GDHs investigated in gram-negative bacteria were PQQ-dependent 23 . GDHs from Bacilli can oxidize a broad range of substrates 24 . The NAD(P) + -GDH from B. thuringiensis oxidizes glucose ( K m 14 mM) and 2-deoxy-D-glucose ( K m 12.2 mM) 17 , and the enzyme from Lysinibacillus sphaericus also oxidizes disaccharides such as maltose, lactose, and sucrose 21 .…”

Enzymes of an Alternative Pathway of Glucose Metabolism in Obligate Methanotrophs

“…To date, microbial NAD(P) + -GDHs have been characterized in gram-positive bacteria, cyanobacteria, archaea, fungi and yeasts, as well as in plants, whereas GDHs investigated in gram-negative bacteria were PQQ-dependent 22 . GDHs from Bacilli can oxidize a broad range of substrates 23 . The NAD(P) + -GDH from B. thuringiensis oxidizes glucose (K m 14 mM) and 2-deoxy-D-glucose (K m 12.2 mM) 17 , and the enzyme from Lysinibacillus sphaericus also oxidizes disaccharides such as maltose, lactose, and sucrose 21 .…”